32

There are many pesticides and insecticides to which pests and insects are

resistant. As a result they are not degraded in the environment by routine processes.

These undegradable compounds however are degradable by bacterial activity (Roberts

et al., 1993). Biodegradation can be defined as the biologically catalyzed reduction in

complexity of chemicals (Michelic and Luthy, 1988). Pesticide degradation in a soil

is a function of multiple factors including population densities and activity of

pesticides degrading microorganisms, pesticide bioavailability and soil parameters

such as pH, soil water content and temperature (Parkin and Daniel, 1994). In soil and

water, pesticides can be degraded by biotic and abiotic pathways; however,

biodegradation by microorganism is the primary mechanism of pesticide breakdown

and detoxification in many soils. Hence, microbes may have a major effect on the

persistence of most pesticides in soil (Surekha Rani et al., 2008).

2.1. Degradation of Chlorpyrifos by bacteria

Rokade and Mali (2013) described the biodegradation of Chlorpyrifos by

Ps. desmolyticum NCIM 2112. The pH and temperature optima for degradation were

found to be 7.0 and at 30°C respectively. Biodegradation was influenced by other

carbon and nitrogen sources and indicated that glucose and maltose were effective at

0.5% concentration and sodium nitrate and yeast extract at 0.05%. Ps. desmolyticum

NCIM 2112 degraded Chlorpyrifos into non toxic metabolites like 2-pyridinol and

thiophosphate.

Lu et al. (2013) isolated and characterized a bacterial strain, Cupriavidus spp.

DT-1 which was capable of degrading Chlorpyrifos and 3, 5, 6-trichloro-2-pyridinol

(TCP). These compounds were used as sole carbon source. The degradation pathway

was investigated and it was shown that Chlorpyrifos was first hydrolyzed to TCP,

33

successively dechlorinated to 2-pyridinol and then subjected to the cleavage of the

pyridine ring and further degradation. Chlorpyrifos-contaminated soil was inoculated

with strain DT-1 which resulted in a degradation rate of Chlorpyrifos and TCP of

100% and 94.3%, respectively as compared to a rate of 28.2% and 19.9% in

uninoculated soil.

From cotton growing areas of Punjab, Farhan et al. (2013) isolated 56

microbial strains capable of Chlorpyrifos degradation. Strain Ct27 (Klebsiella spp.)

was most effective in Chlorpyrifos degradation. Under different conditions like;

concentration, carbon sources, pH and inoculum densities, biodegradation potential of

Klebsiella spp. was studied. Klebsiella spp. showed 90% Chlorpyrifos biodegradation

(200 mg/l) at pH 8 and 105CFU/ml with addition of glucose in 18 days. Chlorpyrifos

degradation was enhanced by the presence of other nutrients probably due to high

growth on easily metabolizable compounds which in turn favoured biodegradation.

This strain could be used for bioremediation and ecological restoration of sites,

contaminated with Chlorpyrifos.

Liu et al. (2012) isolated eleven Chlorpyrifos-degrading bacteria from a

Chinese soil. These strains were significantly different in the ability of degradation

efficiency and one strain, Bacillus cereus, was selected for further analysis. Under

different culture conditions, such as pH, temperature, Chlorpyrifos concentration and

so on, the ability of Bacillus cereus in Chlorpyrifos degradation was investigated.

Bacterial degradation results of Chlorpyrifos in liquid medium indicated that it was

ideally degraded under the condition of 30°C, pH 7.0, concentration of Chlorpyrifos

below 150 mg /1, with a degradation rate up to 78.85%.

34

From the paddy fields of Annamalai Nagar having a history of repeated

pesticide applications, KaviKarunya and Reetha (2012) isolated pesticide degrading

bacteria. The bacterial isolates were identified as Pseudomonas fluorescens, Bacillus

subtilis and Klebsiella spp. Two different pesticides like Monocrotophos and

Chlorpyrifos were used in this study. Klebsiella spp. utilized the pesticides effectively

and showed maximum growth among the three bacterial isolates. In Minimal salt

broth containing 50 ppm of pesticides at different temperature levels like, 25°C, 35°C,

45°C, 55°C and different pH levels like, pH 4, pH 5, pH 6, pH 7, pH 8 and different

carbon sources like, dextrose, fructose, lactose, galactose, mannose and different

nitrogen sources like, malt extract, peptone, yeast extract, casein, beef extract

respectively, the growth of the three pesticide degrading isolates, Pseudomonas

fluorescens, Bacillus subtilis and Klebsiella spp. was assessed. At 35°C and pH 6,

maximum growth rate of bacteria was recorded. In the presence of dextrose, the

growth of bacteria was maximum, followed by fructose, lactose and galactose and the

least growth was recorded in mannose. Maximum growth of bacteria was obtained in

the presence of malt extract followed by peptone, yeast extrac t and casein and the

least growth was recorded in beef extract. Maximum growth of bacteria was recorded

in the Minimal salt broth containing Chlorpyrifos followed by Monocrotophos.

Fifty percent biodegradation of Chlorpyrifos with initial concentration of 480

mg/Kg, by using biobeds or biomixtures (mixture of peat: soil: straw in proportion of

25:25:50 by weight) was recorded by Tortella et al. (2012). Pesticide retention in

biobed depended on the sorption capacity and degradation depended on the biologica l

activity. It was concluded that higher maturity lead to greater degradation.

35

Combined use of plants and microbes for Chlorpyrifos biodegradation was

investigated by Ahmad et al. (2012). The plant and microbes used were rye grass and

Bacillus cereus. The combination of plant and microbes degraded 97% of

Chlorpyrifos within 45 days in soil experimentation. The fact that exogenous

microbes could be used successfully for the bioremediation process was highlighted

by this study.

Pseudomonas spp. from industrial drain was isolated by Farhan et al. (2012).

Good degradation efficiency was shown by this strain as compared to the control.

Complete biodegradation yielded carbon source and energy by the process of

oxidation which was used for the growth of microbes.

Nine morphologically different bacterial strains, one actinomycete and two

fungal strains from the Chlorpyrifos contaminated soil were isolated by Sasikala et al.

(2012). Four bacterial strains which were more efficient among the isolates were

developed as consortium. The four bacterial isolates namely Ps. putida (NII 1117),

Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Ps. aeruginosa (NII

1120) present in the consortia were identified on the basis of 16S R DNA analysis. At

neutral pH and temperature 37°C with Chlorpyrifos concentration 500 mg/l, the

degradation studies were carried out. The presence of metabolites Chlopyrifos oxon

and Diethylphosphorothioate were shown by LC-mass spectral analysis. An important

potential use of this consortium for the cleanup of Chlorpyrifos contaminated

pesticide waste in the environment was highlighted by these results.

Awad et al. (2011) isolated five bacterial isolates (B-CP5- B-CP6 - B-CP7- B-

CP8- B-CP9) from pesticides contaminated soil in Egypt. The isolates were identified

as Pseudomonas stuzeri, Enterobacter aerogenes, Pseudomonas pseudoalcaligenes,

36

Pseudomonas maltophila and Pseudomonas vesicularis respectively based on their

morphological, cultural and biochemical characters. The most potent degrader strain

was Pseudomonas stuzeri which could be used to clean up the areas contaminated

with Chlorpyrifos. When the Pseudomonas stutzeri (B-CP5) was grown on mineral

salts medium supplemented with 0.3 ml/l Chlorpyrifos as sole source of carbon and

energy it could grow within 7 days as optimum incubation period, with preferred

Chlorpyrifos concentration 0.1-0.35 ml/l, maximum inoculum size (0.5 ml), optimum

temperature (30°C), optimum pH (7), most preferred sugar fructose and preferred

nitrogen source ammonium nitrate under shaking condition of 100 rpm. Below and

above optimal incubation period, Chlorpyrifos concentration, inoculum size,

temperature, pH, the phenolic compounds production decreased gradually. Only

fructose could induce biodegradation of Chlorpyrifos whereas all other tested carbon

sources failed to induce the production of phenolic compounds. Ammonium nitrate

was the most preferred nitrogen source whereas other tested nitrogen sources failed to

aid in biodegradation of Chlorpyrifos.

A Chlorpyrifos-degrading strain of Stenotrophomonas acidaminiphila, lux-β

was studied by Wang et al. (2011). The survival and Chlorpyrifos-degrading activity

of strain lux-β in the soil with different treatments were investigated. Colonization and

degrading activity of Chlorpyrifos-degrading bacteria in natural environment was

monitored. It was indicated by results that the quantity of lux-β in the soil rose first

and decreased slowly in sterilized soil, while quickly in non-sterile soil, which

suggested that native microbes had a significant influence on alien microbes. The

quantity of lux-β was larger in soils with Chlorpyrifos than those in soils without

Chlorpyrifos which indicated that Chlorpyrifos could be used as a nutrient by strain

lux-β. Microbes played an important role in degrading Chlorpyifos, and lux-β had

37

higher degradation ability than native microbes, the two of which could generate

synergistic effect on degrading Chlorpyrifos.

Paracoccus spp. strain TRP isolated from activated sludge, by Li et al. (2011),

could completely biodegrade Chlorpyrifos and 3, 5, 6-trichloro-2-pyridinol. The draft

genome of sequence of the strain reported by them could be used to predict genes for

xenobiotic biodegradation and metabolism.

Maya et al. (2011) investigated the efficacy of soil bacterial communities

comprising seven different isolates for biodegradation of Chlorpyrifos and TCP, a

degradation product of Chlorpyrifos. Chlorpyrifos concentration ranged from 25 to

200 mg/l and that of TCP ranged from 25-100 mg/l. The results indicated a high

affinity of bacterial community for degradation of both Chlorpyrifos and TCP. The

genetic relatedness of the isolates 1-4 with Pseudomonas spp., isolates 5 and 6 with

Agrobacterium spp. and isolate 7 with Bacillus spp. was confirmed with 16s rRNA

gene sequence analysis. Their degradation potential for Chlorpyrifos and TCP was

found to be in the order Pseudomonas>Agrobacterium>Bacillus. All seven isolates

were found to be more efficient in degrading TCP compared to Chlorpyrifos.

Kumar et al. (2011a) investigated the Chlorpyrifos degrading capability of

four bacterial monocultures (RCC-2, GCC-1, GCC-3 and JCC-3) and two bacterial

mixed-cultures (GCE345 and GCC134) in terms of treatment duration and culture

volume, using soil slurry medium. RCC-2 was found to be most efficient among the

bacterial mono-cultures with 21, 37, 54 and 77% of Chlorpyrifos degradation in 5, 10,

15 and 30 days of treatment duration, respectively. GCC134 was more effective out of

two bacterial mixed-cultures, and resulted in 24, 38, 56 and 85% degradation of

Chlorpyrifos in 5, 10, 15 and 30 days of treatment, respectively.

38

A strain ZHU-1 capable of utilizing Chlorpyrifos as the sole carbon sources

and energy was isolated by Zhu et al. (2010) from soil. Based on analysis of

morphology, physiological and biochemical characters and 16S rRNA ZHU-1 was

identified as Bacillus licheniformis. The addition of ZHU-1 to soil treated with

Chlorpyrifos resulted in a higher degradation rate than noninoculated soils, the

degradation rate of Chlorpyrifos (100 mg kg-1) could reach 99% or above after 14

days. The microbial manure added by strain ZHU-1 could be applied not only as

fertilizer, but also in degrading Chlorpyrifos residue in soil.

A bacterial strain C2A1was isolated by Anwar et al. (2009) from soil, which

was found to be highly effective in degrading Chlorpyrifos and its first hydrolysis

metabolite 3, 5, 6-trichloro-2-pyridinol (TCP). Strain C2A1 was identified as Bacillus

pumilus. Strain C2A1 utilized Chlorpyrifos as the sole source of carbon and energy.

It was also co-metabolized in the presence of glucose, yeast extract and nutrient broth.

At high pH 8.5 and high inoculum density when Chlorpyrifos was used as the sole

source of carbon and energy, maximum pesticide degradation was observed.

Chlorpyrifos degradation was enhanced in the presence of other nutrients, probably

due to high growth on easily metabolizable compounds which in turn increased

degradation. Within 8 days of incubation the strain C2A1 showed 90% degradation of

TCP (300 mg/l).

By selective enrichment technique, JayaMadhuri and Rangaswamy (2009)

isolated Bacillus spp. and Pseudomonas spp. These isolates were tested for their

ability to degrade the respective insecticides in mineral salts medium. Nearly 75% of

Chlorpyrifos and phorate and 50% of Dichlorvos, Methyl parathion and Methomyl

were degraded by cultures of soil bacteria within 7 days of incubation. Formation of

39

one unidentified metabolite in inoculated samples was revealed by qualitative analysis

of Chlorpyrifos and Methyl parathion residues by gas chromatography; whereas no

metabolite formation was detected in case of other insecticides inoculated samples.

Dichlorvos and Phorate were completely degraded by the soil isolates at the end of

14 days. It was observed that Phorate > Dichlorvos > Methyl parathion >

Chlorpyrifos> Methomyl; was the order of microbial degradation of selected

insecticides in the experiment.

Kim and Ahn (2009) isolated a Chlorpyrifos-methyl (CM) degrading bacterial

strain KR100 from a Korean rice paddy soil. This bacterium showed greatest

similarity to members of the order Burkholderiales. Based on morphological,

biochemical, and molecular characteristics it was shown to be most closely related to

members of the Burkholderia cepacia group. CM was hydrolyzed to 3, 5, 6-trichloro-

2-pyridinol (TCP) by strain KR100. TCP was utilized by the bacterium as the sole

source of carbon for its growth. The isolate was also able to degrade Chlorpyrifos,

Dimethoate, Fenitrothion, Malathion and Monocrotophos at 300 μg/ml but Diazinon,

Dicrotophos, Parathion, and Parathion-methyl at 100 μg/ml. The ability to degrade

CM was found to be encoded on a single plasmid of ~50 kb, pKR1.

From an agricultural soil, Singh et al. (2009) isolated a Pseudomonas spp. by

enrichment culture technique in the presence of Chlorpyrifos. ChlD was capable of

producing biosurfactant (rhamnolipids) and degrading Chlorpyrifos (0·01 g/l). The

evaluation of the partially purified rhamnolipid biosurfactant preparation, having a

CMC of 0·2 g/l, for its ability to enhance aqueous phase partitioning and degradation

of Chlorpyrifos (0·01 g/l) by ChlD strain was carried out. It was validated by GC and

HPLC studies that at 0·1 g/l supplement of biosurfactant the best degradation

40

efficiency was observed. More than 98% degradation of Chlorpyrifos was obtained

with the addition of biosurfactant at 0·1 g/l when compared to 84% in the absence of

biosurfactant after 120 h of incubation.

From waste waters of chemical factories and contaminated agricultural soil,

Zanjani et al. (2009) isolated ten bacterial strains using Chloropyrifos and Diazinon as

source of carbon and phosphorus. Among these, IHU strain4; grew most rapidly and

luxuriously and showed the highest organophosphate-hydrolyzing capability. The

bacterial isolate was identified as a member of the genus Pseudomonas on the basis of

morphological and biochemical characteristics.

Vidya Lakshmi et al. (2009) developed three aerobic bacterial consortia, AC,

BC and DC, from pesticide-contaminated soils of Punjab which were able to degrade

Chlorpyrifos after 21 days of incubation in basal medium by 54, 46 and 61% and

Chlorpyrifos in soil after 30 days by 50, 56, and 64%. From these consortia

Ps. aeruginosa, Bacillus cereus, Klebsiella spp., and Serratia marscecens were

obtained which showed 84, 84, 81, and 80% degradation of Chlorpyrifos in liquid

medium after 20 days and 92, 60, 56 and 37% degradation of Chlorpyrifos in soil

after 30 days. In soil experiments, populations of Bacillus cereus, Klebsiella spp. and

Serratia marscecens remained steady. The only exception observed was with P.

aeruginosa, where the population showed a substantial increase. It was observed

during the degradation of Chlorpyrifos by Ps. aeruginosa, that 3, 5, 6-trichloro-2-

pyridinol, the major metabolite of Chlorpyrifos degradation was formed which

disappeared to negligible amounts.

Fang et al. (2009) investigated the degradation of Chlorpyrifos at different

concentrations in soil. Its impact on soil microbial functional diversity under

41

laboratory condition was studied. It was calculated that the degradation half- live of

Chlorpyrifos at levels of 4, 8, and 12 mg/kg in soil were 14.3, 16.7, and 18.0 days,

respectively. It was concluded that Chlorpyrifos residues in soil had a temporary or

short-term inhibitory effect on soil microbial functional diversity.

Seven Chlorpyrifos-degrading bacteria, named Dsp-1 to Dsp-7 were isolated

by Li et al. (2008) from organophosphate pesticide-contaminated soil and water using

a culture method with Chlorpyrifos as the sole carbon source. In order to study their

morphological, physiological and biochemical characteristics comparative studies

were performed. Strains Dsp-2, Dsp-4, Dsp-6 and Dsp-7 were identified as

Sphingomonas spp., Stenotrophomonas spp., Bacillus spp. and Brevundimonas spp.,

respectively based on these analyses, while all other strains were members of

Pseudomonas spp. It was shown by the results that there were diversities of

Chlorpyrifos-degrading strains in the contaminated environment, and the

Chlorpyrifos-degrading strains had the potential to clean up the organophosphate

pesticide-contaminated environment.

The bioremediation potential of Ps. aeruginosa (NCIM 2074) was assessed by

Fulekar and Geetha (2008) by improving its adaptability to increasing concentration

of Chlorpyrifos using scale up process. By subjecting to varying concentrations of

Chlorpyrifos, i.e. 10, 20, 50, 75 and 100 mg/l Ps. aeruginosa isolate NCIM 2074 was

adapted. The entire scale up process continued for a period of 70 days. To increasing

concentrations of Chlorpyrifos upto 50 mg/l, Ps. aeruginosa (NCIM 2074) was

adapted, but 75 and 100 mg/l concentrations were inhibitory to the organism. GC-MS,

showed that biodegradation of Chlorpyrifos at 10, 25, 50 mg/l degraded completely

over a period of 1, 5 and 7 days, respectively. It was shown by GC/MS that

42

Chlorpyrifos was degraded upto 52% in Mineral salts medium (MSM) containing 75

mg/l Chlorpyrifos while in MSM containing100 mg/l Chlorpyrifos it was degraded

upto 25%. Chlorpyrifos was completely degraded in the rest of the concentrations that

were evaluated (Geetha, 2008). During bioremediation, the intermediate 3, 5, 6

trichloro-2-pyridion, 2, 4-bis (1, 1 dimethyiethyl) phenol and 1, 2

benzenedicarboxylic acid persisted, but in the long run these were converted to CO2,

biomass and nutrients.

Four different bacterial strains were isolated by Surekha Rani et al. (2008)

from agricultural soil. Three isolates among them were shown to belong to the family

Enterobacteriaceae and one isolate belonged to the genus Bacillus. The isolate

selected for further characterization in this study, MS09, showed the greatest

similarity to members of the order Enteriobacteriales and, in particular, to the

Providencia genus. MS09 was identified and characterized as a strain of Providencia

stuartii. By selective enrichment on mineral medium containing Chlorpyrifos, MS09

was capable of utilizing Chlorpyrifos as sole carbon source. It was shown by growth

studies that MS09 utilized Chlorpyrifos to grow in Luria-Bertani broth containing

different concentrations of Chlorpyrifos at 50 -700 mg/l.

Vidyalakshmi et al. (2008) developed five aerobic consortia capable of

degrading Chlorpyrifos as a sole carbon source in aqueous medium which showed

degradation in the range of 46–72% after 20 days. From these consortia,

Pseudomonas fluorescence, Brucella melitensis, Bacillus subtilis, Bacillus cereus,

Klebsiella species, Serratia marcescens and Pseudomonas aeroginosa, were isolated

which showed 75–87% degradation of Chlorpyrifos as compared to 18% in control

after 20 days of incubation. Bioremediation of Chlorpyrifos-contaminated soil with

43

Ps. fluorescence, B. melitensis, B. subtilis and Ps. aeroginosa individually showed

89%, 87%, 85% and 92% degradation, respectively, as compared to 34% in control

after 30 days. 3,5,6-trichloro-2-pyridinol (TCP) was detected as metabolite of

Chlorpyrifos degradation by Ps. aeroginosa after 20 days, during bioremediation

studies, which was utilized and disappeared after 30 days. By whole-cell studies it

was shown that Ps. aeroginosa gave TCP as the product of Chlorpyrifos degradation,

which was further metabolized to unknown polar metabolites.

Xu et al. (2008) isolated a bacterial strain TRP, from activated sludge which

could biodegrade Chlorpyrifos and 3, 5, 6-trichloro-2-pyridinol. With the study of

phenotypic features, physiological and chemotaxonomic characteristics, and

phylogenetic analysis of 16S rRNA sequence it was shown that the isolate belonged

to the genus Paracoccus. When provided as sole carbon and energy sources, pyridine,

methyl parathion and carbonfuran could also be degraded by strain TRP. Involvement

of an alternative degradation mechanism with an inducible enzyme was indicated by

Native-PAGE and enzymatic degradation assay of the cell- free extracts. No persistent

accumulated metabolite was observed when degradation of Chlorpyrifos by strain

TRP was examined by GC–MS and HPLC. It was observed that this was the first

report of a bacterium that could completely mineralize Chlorpyrifos. This isolate

showed that it would be potentially useful in biotreatment of wastewaters and

bioremediation of contaminated soils.

Four bacterial isolates designated as RA-3, RA-5, RA-10, RA-20, were

isolated from waste water irrigated agricultural soil with previous history of

Chlorpyrifos use by Bhagobaty and Malik (2008) using enrichment culture technique.

These showed promising capability of utilizing Chlorpyrifos as a carbon source for

44

their growth. Morphological and biochemical tests performed on the bacteria

indicated that they might belong to the genus Pseudomonas. The degradation of

Chlorpyrifos by these strains was shown by Thin layer chromatography and

tetrazolium reduction assay.

From the polluted treatment system of a Chlorpyrifos manufacturer, Li et al.

(2007b) isolated a highly effective Chlorpyrifos-degrading bacterium strain Dsp-2

which was preliminarily identified as Sphingomonas spp. By hydrolyzing

Chlorpyrifos to 3, 5, 6-trichloro-2-pyridinol (TCP), it utilized Chlorpyrifos as its sole

source of carbon for growth. When bioremediation of Chlorpyrifos-contaminated soil

was examined using Dsp-2, its addition to soil treated with 100 mg/ kg Chlorpyrifos

resulted in a higher degradation rate than control soils without inoculation. The

moderate pH, moisture and inoculum density could have promoted degradation.

Ghanem et al. (2007) isolated a Chlorpyrifos (CPY)-degrading bacterial strain

from an activated sludge sample collected from the Damascus Wastewater Treatment

Plant, Syria. The addition of CPY at a rate of 3.84 g/l of sludge weekly facilitated the

isolation of this Klebsiella spp. Three activated sludge samples were incubated in the

presence of CPY (13.9 g/l sludge). Within 4 days 46% of added CPY was degraded.

By comparison, the isolated Klebsiella spp. was found to breakdown 92% of CPY

when co- incubated in a poor mineral medium in which CPY was the sole carbon

source (13.9 g/l poor medium) within 4 days. In the poor medium isolated Klebsiella

spp. was able to tolerate up to 17.3 g of CPY.

Xu et al. (2007) isolated a bacterial strain, Serratia spp. that could transform

Chlorpyrifos to 3, 5, 6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal

strain, Trichosporon spp. from an activated sludge by enrichment culture technique.

45

The fungus could also degrade 50 mg Chlorpyrifos/l within 7 days. Complete

mineralization of 50 mg Chlorpyrifos/l within 18 h at 30 °C and pH 8 using a total

inoculum of 0.15 g biomass/l was observed when co-cultures were used.

Yang et al. (2006) isolated an effective Chlorpyrifos-degrading bacterium,

strain YC-1 from the sludge of the wastewater treating system of an

organophosphorus pesticides manufacturer. It was identified as the genus

Stenotrophomonas based on the results of phenotypic features, phylogenetic similarity

of 16S rRNA gene sequences and BIOLOG test. Chlorpyrifos was degraded to 3, 5,

6-trichloro-2-pyridinol when it was utilized as the sole source of carbon and

phosphorus for growth by YC-1. When provided as the sole source of carbon and

phosphorus, strain YC-1, was also able to degrade Parathion, Methyl parathion, and

Fenitrothion. When YC-1 (106 cells g/1) was inoculated into soil treated with 100 mg/

kg Chlorpyrifos, it resulted in a higher degradation rate than in non inoculated soils.

It was found by Singh et al. (2006) that both, Chlorpyrifos-degrading

Enterobacter spp. and fenamiphos-degrading consortium rapidly degraded pesticides

when inoculated into natural and sterile water and soils. In comparison with natural

and alkaline soils, degradation rate was slower in soils with lower pH. It was observed

that soil organic matter had no impact on pesticide degrading ability of isolates.

Degradation rate was slowed down by soil moisture <40% of maximum water-

holding capacity. Rapid degradation of fenamiphos and Chlorpyrifos by the bacterial

isolates was observed between 15°C and 35°C. Their degradation ability was sharply

reduced at 5°C and 50 °C. Both groups of bacterial systems were also able to remove

a range of pesticides by degradation. For initiating rapid growth and degradat ion, an

inoculum density of 104 cells g/l of soil was required. Contrasting results were

46

produced by ageing of pesticide in soils prior to inoculation. Ageing of fenamiphos

had no impact on subsequent degradation by the inoculated consortium whereas,

degradation of Chlorpyrifos by Enterobacter spp after aging resulted in persistence of

10% of pesticide in soil matrix. Under different soil and water characteristics the

bioremedial potential of a fenamiphos degrading consortium and a Chlorpyrifos

degrading bacterium was confirmed by these studies.

Twenty Chlorpyrifos resistant bacterial isolates were isolated, identified and

characterized by Ajaz et al. (2005) from cotton cultivated soil of NIAB, Faisalabad,

Pakistan using conventional and API kit methods. Among these, four were gram

positive cocci; three were gram positive bacilli while 13 were gram negative bacilli.

For further studies, three Chlorpyrifos hyper resistant bacteria were finally selected.

Three isolates, Ps. putida, Aeromonas spp., and Klebsiella spp., were found to resist 2

mg/ml, 4 mg/ml and 8 mg/ml of Chlorpyrifos while Ps. putida and Aeromonas spp.,

also resisted the 10 mg/ml and 20 mg/ml doses.

Yang et al. (2005) isolated a bacterium DSP3, from contaminated soils around

a chemical factory which was capable of degrading Chlorpyrifos and 3, 5, 6-trichloro-

2-pyridinol. Strain DSP3 was identified as Alcaligenes faecalis based on the results of

phenotypic features, phylogenetic similarity of 16S rRNA gene sequences, DNA G+C

content and DNA homology between strain DSP3 and reference strains. Chlorpyrifos

was utilized as the sole source of carbon and phosphorus by DSP3. Chlorpyrifos and

3, 5, 6-trichloro-2-pyridinol degradation by DSP3 under different culture conditions

was examined. When provided as the sole sources of carbon and phosphorus,

parathion and diazinon could also be degraded by strain DSP3. An addition 108 cells/g

of DSP3 to soil with Chlorpyrifos (100 mg/kg) resulted in a higher degradation rate

47

than the one obtained from non-inoculated soils. In six types of treated soils, different

degradation rates of Chlorpyrifos were observed. It was suggested that soils used for

growing cabbage in combination with inoculation of strain DSP3 showed enhanced

microbial degradation of Chlorpyrifos.

Six Chlorpyrifos-degrading bacteria were isolated by Singh et al. (2004) from

an Australian soil and compared by biochemical and molecular methods. Among

these isolates which were indistinguishable, one strain, B-14 was selected for further

analysis which showed greatest similarity to members of the order Enterobacteriales

and was closest to members of the Enterobacter asburiae group. Under different

culture conditions, the ability of the strain to mineralize Chlorpyrifos was investigated

and the strain utilized Chlorpyrifos as the sole source of carbon and phosphorus.

Hydrolysis of Chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-

pyridinol and utilization of DETP for growth and energy by the isolate was

demonstrated by studies with ring or uniformly labeled [14C] Chlorpyrifos in liquid

culture. Mono- and diphosphatase activities along with a phosphotriesterase activity

were possessed by the isolate and it degraded the DETP-containing organophosphates

Parathion, Diazinon, Coumaphos and Isazofos when provided as the sole source of

carbon and phosphorus. The isolate could not degrade Fenamiphos, Fonofos,

Ethoprop and Cadusafos, which have different side chains. When the strain B-14

(106 cells g/1) was added to soil with a low indigenous population of Chlorpyrifos

degrading bacteria treated with 35 mg of Chlorpyrifos kg/l it resulted in a higher

degradation rate than was observed in noninoculated soils. The potential of this

bacterium to be used in the cleanup of contaminated pesticide waste in the

environment was highlighted by these results.

48

Singh et al. (2003) examined the role of microorganisms in the degradation of

the organophosphate insecticide Chlorpyrifos in soils from the United Kingdom and

Australia. Dissipation of Chlorpyrifos mediated by the cometabolic activities of the

soil microorganisms was suggested by the kinetics of degradation in five United

Kingdom soils varying in pH from 4.7 to 8.4. It was observed that repeated

application of Chlorpyrifos to these soils did not result in the development of a

microbial population with an enhanced ability to degrade the pesticide. In an

Australian soil a robust bacterial population that utilized Chlorpyrifos as a source of

carbon was detected and the enhanced ability to degrade Chlorpyrifos in the

Australian soil was successfully transferred to the five United Kingdom soils. This

degrading ability after 90 days of inoculation was maintained only in soils with a pH

of >6.7.

2.2. Degradation of Chlorpyrifos by fungi and actinomycetes

According to Yu et al. (2006) fungi possess the biochemical and ecological

capacity to detoxify environmental xenobiotics, either by chemical modification or by

influencing chemical bioavailability. Moreover, it was suggested by Harms (2011)

that the ability of fungi to form extended mycelial networks, the low specificity of

their catabolic enzymes and their independence from utilizing organic chemicals as a

growth substrate make fungi well suited for bioremediation processes.

Fungal isolates were obtained by Maya et al. (2012) from soils; which were

then used for degrading Chlorpyrifos (CP) and 3, 5, 6- Trichloro-2-pyridinol (TCP).

For Chlorpyrifos, the percentage degradation ranged from 69.4 to 89.8 and that for

TCP ranged from 62.2 to 92.6 after one week. Ks and Vmax values were different for

different isolates. The Ks ranged from 66.66 to 169.5 mg/l and Vmax from 6.56 to 40.4

49

mg/l/d for CP and from 53.19 to 163.9 mg/l and 3.41 to 40.40 mg/l/d, respectively, for

TCP. High affinity for both CP and TCP was shown by the fungal community. The

genetic relatedness of isolate F1 to Aspergillus sp., F2 and F3 to Penicillium sp., F4 to

Eurotium sp. and F5 to Emericella sp. were confirmed. The degradation potential was

in the order: F1>F2=F3>F4>F5.

The actinomycete strain Streptomyces radiopugnans and two fungal strains

Aspergillus niger, Tricophyton spp. were isolated by Sasikala et al. (2012) from a

Chlorpyrifos contaminated soil.

Chen et al. (2012) isolated a new fungal strain Hu-01 with high Chlorpyrifos-

degradation activity. Based on the morphology and 5.8S rDNA gene analysis Hu-01

was identified as Cladosporium cladosporioides which utilized 50 mg/l of

Chlorpyrifos as the sole carbon of source and tolerated high concentration of

Chlorpyrifos up to 500 mg/l. Based on the Response Surface Methodology (RSM)

26.8°C and pH 6.5 were found to be the optimum conditions for degradation, under

which Hu-01 completely metabolized the supplemented Chlorpyrifos (50 mg/l) within

5 days. Transient accumulation of 3, 5, 6-trichloro-2-pyridinol (TCP) was observed

during the biodegradation process, which did not accumulate in the medium and

disappeared quickly. At the end of experiment no persistent accumulative metabolite

was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis. The

metabolic pathway for the complete detoxification of Chlorpyrifos and its hydrolysis

product TCP was harboured by the isolate, suggesting that the fungus may be a

promising candidate for bioremediation of Chlorpyrifos-contaminated water, soil or

crop.

50

By enrichment technique Kulshrestha and Kumari (2011) isolated mixed and

pure fungi from three soils. When cultivated in Czapek Dox medium the enriched

mixed fungal cultures were capable of biodegrading Chlorpyrifos (300 mg/l). The

pure fungal strain, identified as Acremonium spp., utilized Chlorpyrifos as a source of

carbon and nitrogen. Acremonium spp. strain GFRC-1 showed highest Chlorpyrifos

degradation of 83.9% when cultivated in the nutrient medium with full nutrients. The

major biodegradation product of Chlorpyrifos which was detected was Desdiethyl

Chlorpyrifos. This study indicated that isolated fungal strain would be used for

developing bioremediation strategy for Chlorpyrifos-polluted soils.

Fang et al. (2008) investigated the degradation characteristics of Chlorpyrifos

by an isolated fungal strain Verticillium spp. DSP in pure cultures, soil and on pakchoi

(Brassica chinensis L.). The rate of degradation of Chlorpyrifos in the mineral salts

medium was proportional to the concentrations of Chlorpyrifos ranging from 1 to

100 mg/l. The rate of degradation for Chlorpyrifos (1 mg/l) in the mineral salts

medium was 1.12 and 1.04 times faster at pH 7.0 than those at pH 5.0 and 9.0 and the

degradation at 35°C was 1.15 and 1.12 times faster, respectively, than those at 15 and

20 °C. The degradation of Chlorpyrifos was increased significantly by the addition of

the fungal strain DSP into the contaminated soils. The rates of Chlorpyrifos

degradation in inoculated soils were 3.61, 1.50 and 1.10 times faster in comparison

with the sterilized soil, previously Chlorpyrifos-untreated soil and previously

Chlorpyrifos-treated soil under laboratory conditions. In contrast to the controls, the

half- lives of Chlorpyrifos were significantly shortened by 10.9% and 17.6% on

treated pakchoi, 12.0% and 37.1% in inoculated soils, respectively, in the greenhouse

and open field. The fact that the fungal strain DSP could be used successfully for the

51

removal or detoxification of Chlorpyrifos residues in/on contaminated soil and

vegetable was indicated by these results.

Yu et al. (2006) by enrichment cultivation isolated a fungal strain DSP from

soil, capable of utilizing Chlorpyrifos as sole carbon and energy sources. In mineral

salt medium, 2.03, 2.93 and 3.49 days, were the half- lives of degradation (DT50) for

Chlorpyrifos at concentrations of 1, 10, and 100 mg/l, respectively by DSP. To

enhance Chlorpyrifos degradation on vegetables, two cell- free extracts [E (1:10) and

E (1:20)] from the fungal strain DSP in bran–glucose medium were prepared. The

DT50 of Chlorpyrifos were reduced by 70.3%, 65.6%, 80.6%, 80.6% and 86.1%, and

by 53.8%, 43.2%, 66.0%, 54.3% and 67.7% on E (1:20) and E (1:10) treated pakchoi,

water spinach, Malabar spinach, haricot beans, and pepper, respectively, compared

with the controls. The 7-day residual values (R 7) of Chlorpyrifos on E (1:10) treated

vegetables were all lower than the corresponding maximum residue levels of

European Union (EU MRLs), except that the R 7 value on haricot beans was slightly

higher than the corresponding EU MRLs. By the results obtained, it was indicated that

cell- free extracts could rapidly degrade Chlorpyrifos residues on vegetables.

From the groundnut fields in Rajasthan, Swati and Singh (2002) isolated

fungal strains Aspergillus niger and A. flavus which utilized Chlorpyrifos as a sole

source of carbon and phosphorus. A. flavus utilized about 96.2% of Chlorpyrifos as

nitrogen and phosphorus source within 24 hours of incubation when it was provided

with 200 mg/Kg of Chlorpyrifos. A. niger reduced the concentration of Chlorpyrifos

to 95.6 mg/Kg within 24 hours of inoculation by utilizing 52.2% of the total

Chlorpyrifos.

52

Bending et al. (2002) reported the degradation of Chlorpyrifos in ‘biobed’

composting substrate by two white-rot fungi, Hypholoma fascicularae and Coriolus

versicolor. Chlorpyrifos was hydrolyzed and the pyridinyl ring further underwent

cleavage before being converted to carbon dioxide and water.

Al-Mihanna et al. (1998) studied biodegradation of Chlorpyrifos in liquid

culture media amended with either single or combined eight different plant pathogenic

fungi isolated from the continuous cropping wheat fields. From the liquid media the

average recovery of Chlorpyrifos was found to be 86.1%. At Chlorpyrifos

concentrations up to 200 ppm growth of mixed fungi was higher than in the control

treatment. Concentrations of Chlorpyrifos declined in the medium of combined fungi

more than it did in the medium of any single fungus with increase in the incubation

period. After 21 days, 79.8 ppm (39.9%) of Chlorpyrifos was recovered in the

combined fungal cultures. From the media with Fusarium graminearum,

F. oxysporum, Rhizoctonia solani, Cladosporium cladosporiodes, Cephalosporium

spp., Trichoderma viridi, Alternaria alternata, and Cladorrhinum brunnescens, 48.0

to 74.8% of Chlorpyrifos was recovered. In the medium amended with mixed fungi

the half‐life value (T1/2) for Chlorpyrifos was 15.8 days. It ranged from 19.3 to 33.0

days for the single cultures.

2.3. Degradation of Endosulfan by bacteria

Vijaiyan Siva and Rajam (2013) isolated a bacterium capable of degrading

Endosulfan which was found to be tolerant to 250 ppm of Endosulfan. It was

indicated that bacterium belonged to Pseudomonas spp. by the 16s rRNA sequence

analysis. When the medium was inoculated with Pseudomonas spp. ED1, thin layer

chromatography (TLC) analysis showed the disappearance of β-Endosulfan and

53

formation of a new metabolite. Presence of Endosulfan monoaldehyde was shown by

Gas chromatography-mass spectrum (GC-MS) analysis.

Kong et al. (2013) isolated Endosulfan-degrading bacterial strain, Alcaligenes

faecalis JBW4 from activated sludge which was able to use Endosulfan as a carbon

and energy source. Identification of the optimal conditions for the growth of strain

JBW4 and for biodegradation by this strain was done. The metabolic products of

Endosulfan degradation were studied in detail. Endosulfan biodegradation by strain

JBW4 occurred maximally using a broth with an initial pH of 7, an Endosulfan

concentration of 100 mg/l and incubation temperature of 40°C. Using gas

chromatography (GC) the concentration of Endosulfan was determined. 87.5% of

α-Endosulfan and 83.9% of β-Endosulfan was degraded by JBW4 within 5 days.

Major metabolites detected by gas chromatography-mass spectrometry (GC-MS) were

Endosulfan diol and Endosulfan lactone. Persistent and toxic metabolite, Endosulfan

sulfate, was not detected. Degradation of Endosulfan by A. faecalis JBW4 via a non-

oxidative pathway was suggested by these results. This was the first report on the

biodegradation of Endosulfan by A. faecalis. Potential use of JBW4 for the

bioremediation of environments that are contaminated with Endosulfan residues was

indicated by the results.

To isolate bacteria capable of producing biosurfactant that solublizes

Endosulfan and for enhanced degradation of Endosulfan and its major metabolite

endosulfate a study was carried out by Odukkathil and Vasudevan (2013). As the

degradation of Endosulfan is limited due to its low solubility, the significance of the

study was to enhance the bioavailability of soil-bound Endosulfan residues. From a

pesticide-contaminated soil, a mixed bacterial culture capable of degrading

54

Endosulfan was enriched which was able to degrade about 80% of α-Endosulfan and

75% of β-Endosulfan in five days. Screening of bacterial isolates for biosurfactant

production and Endosulfan degradation was carried out. Four strains, among the

isolates screened, produced biosurfactant on Endosulfan. Better emulsification of

Endosulfan and 99% degradation of Endosulfan and 94% degradation of endosulfate

formed during Endosulfan were shown by ES-47. Reduction of surface tension up to

37 dynes/cm was achieved by the strain. It was revealed by the study that the strain

was capable of degrading Endosulfan and endosulfate with simultaneous biosurfactant

production.

Neelambari and Annadurai (2013) studied the degradation and detoxification

potential of Endosulfan by halotolerant bacterial strains. Streptococcus agalactiae a

halotolerant Endosulfan degrading bacterium which tolerate upto 5000 ppm of

Endosulfan was isolated from enriched sediment of the Vellar estuary after repeated

screening. Endosulfan degradation efficiency of 40.77% was shown by GC-MS

analysis with 80% chloride release in the medium. pH 8, 35˚C, dextrose as carbon

source, salinity 30 ppt inoculum size of 100 μl/ml or 10ml/l broth with 2.5×106

CFU/ml under shaking condition with 15 days incubation were the optimum growth

parameters of S. agalactiae for Endosulfan degradation. It was concluded that this

potential strain could be useful in reclamation of any contaminated site in the tropics.

Reddy et al. (2012) isolated several strains of bacteria and fungi from

Endosulfan contaminated soil. Among the total of 29 bacterial and 16 fungal strains

that were tested for their Endosulfan degradation potential, the disappearance of

Endosulfan from the spiked and inoculated broth varied substantially. The

degradation ranged from 40 to 93% of the spiked amount (100 mg/l) as the strains

55

differed substantially in their potential to degrade Endosulfan in vitro. There was a

substantial decrease in pH of the broth from 8.2 to 3.2 within 14 days of incubation

due to biodegradation of Endosulfan by these microorganisms. Endosulfan diol and

Endosulfan ether were among the products of Endosulfan metabolism by these

microbial strains as revealed by High performance liquid chromatography analyses. A

persistent and toxic metabolite of Endosulfan, Endosulfan sulfate, was not detected in

any case.

Giri and Rai (2012) isolated bacteria from Endosulfan-contaminated soil and

grew the isolates in minimal medium and screened for Endosulfan degradation. For

further studies, the isolate which used Endosulfan and showed maximum growth was

selected. 92.80% of α and 79.35% of β-Endosulfan isomers in 15 days at 20 mg/l

concentration was the maximum degradation in shake flask culture by Pseudomonas

fluorescens, followed by 50 and 100 mg/l while the corresponding values in static

condition were 69.15 and 51.39%, respectively. It was seen that Endosulfan

degradation declined significantly at 50 and 100 mg/l. Release of chloride ion

concomitant to degradation exhibited positive relation. In agitating condition pH

decreased from 7.0 to 4.53 where as in static condition it decreased from 7.0 to 5.18.

Maximum Endosulfan degradation was seen in sterilized soil amended with

Ps. fluorescens as revealed by the soil microcosm study. In broth culture Endosulfan

diol and Endosulfan ether were among the products of Endosulfan metabolism.

Endosulfan ether was the only product detected in the soil microcosm. In either case

Endosulfan sulfate, a persistent and toxic metabolite of Endosulfan, was not detected.

Use of Ps. fluorescens effectively for bioremediation of the pesticide contaminated

sites was indicated by the study.

56

By the process of enrichment, Sunitha et al. (2012a) isolated a bacterial

culture from the soils of an Endosulfan contaminated coffee cultivated area. More

than 70% of Endosulfan and 90% of Endosulfan sulfate degradation was observed

when the culture was grown in the presence of Endosulfan or Endosulfan sulfate as

the sole source of sulfur. Strains of Ps. putida, differing in their 16s rRNA sequence

were found in the culture. In the presence of Endosulfan and Endosulfan sulfate

respectively an increase in biomass up to 1.8 g/l and 2.2 g/l was observed. In the

cultures, metabolites of Endosulfan degradation like, Endosulfan diol, Endosulfan

lactone and Endosulfan sulfate were detected. Compared to static conditions, the

culture showed higher degradation under aerated conditions. It was indicated by this

study that with the ability to tolerate high concentrations of Endosulfan and

Endosulfan sulfate, this culture had the potential for use as a bioremediating

agent for contaminated soil and water.

The aim of the study conducted by Sunitha et al. (2012b) was to delineate

Endosulfan residues in soil, representing samples drawn from agricultural fields

during winter season. After pre-treatment the samples were subjected to enrichment of

the residues. GC-ECD method was used to estimate the residues. 0.1 mg to 29 mg/Kg

was the range of concentration of α-Endosulfan while the levels of β-Endosulfan

ranged from 0.1 to 167 mg/kg and that of Endosulfan sulfate ranged from 0.12 to 27

mg/Kg. Ability of Endosulfan degradation was possessed by the microorganisms

present in this soil by as they were chronically exposed to it. Endosulfan and

Endosulfan sulfate were degraded upto 70% and 100% respectively. Therefore it was

concluded that the bacteria isolated from Endosulfan contaminated soils by the

process of enrichment could be used as bioremediating agents for soil and water.

57

Endosulfan bioremediation metabolizing potential of a bacterial strain

Rhodococcus spp. MTCC 6716, isolated from the gut of an Indian earthworm

(Metaphire posthuma) was studied by Verma et al. (2011). The study included the

optimum conditions for the maximum growth, kinetics of Endosulfan degradation and

half life. In a liquid culture medium at pH 7.0, 30°C and 0.085 M sodium chloride

concentration maximum growth of bacteria was observed. Within 15 days,

Rhodococcus strain degraded Endosulfan up to 97.23% without producing toxic

metabolite with a half life of 5.99 days.

Through selective enrichment technique using sulfur free medium with

Endosulfan as sole sulfur source, Singh and Singh (2011) isolated Endosulfan

degrading bacteria from soil. Strain C8B was found to be the most efficient

Endosulfan degrader, out of the 8 isolated bacterial strains. It degraded 94.12%

α-Endosulfan and 84.52% β-Endosulfan. On the basis of 16S rDNA sequence

similarity strain C8B was identified as Achromobacter xylosoxidans which was also

found to degrade 80.10% Endosulfan sulfate using it as sulfur source. During the

entire course of degradation no known metabolites were found to be formed in the

culture media and the bacterial strain was found to degrade all the known Endosulfan

metabolites. Release of CO2 from the culture media was markedly increased with

Endosulfan as sulfur source as compared to magnesium sulphate. This suggested that

Achromobacter xylosoxidans through the formation of Endosulfan ether, probably

degraded Endosulfan completely.

Through enrichment culture techniques, using technical-Endosulfan, Bajaj et

al. (2010) isolated Pseudomonas spp. strain IITR01 capable of degrading α-ES and

toxic ES sulfate. When β-ES was used, no growth and no degradation were observed.

58

By using the crude cell extract of IITR01, metabolism of α-ES and endosulfate was

also observed. Disappearance of both α-ES and ES sulfate and the formation of

hydroxylated products ES diol, ether and lactone was revealed by thin layer

chromatography and gas chromatography-mass spectrum analysis. Using SDS–

PAGE, the molecular mass of protein induced during the degradation of α-ES and

sulfate as substrate was found to be approximately 150 kDa.

Elsaid et al. (2010) isolated soil microorganisms; bacteria, actinomycetes and

fungi, from highly polluted soils of pesticide stores and cotton field. The effects of

these on half- lives of α and β- Endosulfan under condition of selective and carbon

free media were studied. Significant decrease in half- lives ranging between 58.4 –

81.9% in α-Endosulfan compared to 35.5 –71.6% in β- isomer was observed.

Jayashree and Vasudevan (2009) studied the degradation of alpha and beta

Endosulfan by Ps. aeruginosa with Tween 80 and different moisture regimes (flooded

and non-flooded conditions). Maximum degradation of 92 % with maximum bacterial

count was seen in non-flooded and Tween 80 added soil. Solubility and degradation

of Endosulfan was enhanced by the addition of synthetic surfactant Tween 80. Both

the isomers were degraded with the formation of endodiol and Endosulfan sulfate. It

was concluded that 92% degradation of Endosulfan in contaminated soil was achieved

by Ps. aeruginosa combined with Tween 80.

Goswami and Singh (2009), isolated bacterial strains from Endosulfan treated

soil to study the microbial degradation of this pesticide in broth medium and soil

microcosm. These were grown in minimal medium and screened for Endosulfan

degradation. For detailed studies, the strain, which utilized Endosulfan and showed

maximum growth, was selected. In shake flask culture, Bordetella spp. B9 showed

59

maximum degrading capability. 80% of α-Endosulfan and 86% of β-Endosulfan was

degraded by B9 in 18 days. In broth culture, Endosulfan ether and Endosulfan lactone

were the main metabolites, whereas, in soil microcosm Endosulfan sulfate was also

found along with Endosulfan ether and Endosulfan lactone. Using this strain in six

different treatments, soil microcosm study was also carried out. It was concluded that

this bacterial strain had a potential to be used for bioremediation of the contaminated

sites.

From an activated sludge, Li et al. (2009b) isolated Achromobacter

xylosoxidans CS5, capable of utilizing Endosulfan as the sole carbon, sulfur and

energy source. Using HPLC, degradation of Endosulfan by strain CS5 was examined.

More than 24.8 mg/l α-Endosulfan and 10.5 mg/l β-Endosulfan was degraded after 8

days in aqueous medium with the formation of Endosulfan diol and Endosulfan ether

as the major metabolites; this was demonstrated by analysis of culture pH, cells

growth and residual Endosulfan. Metabolism of Endosulfan was rapid by cell- free

extract of strain CS5 and the degradative enzymes were constitutively expressed.

Removal of Endosulfan in soil was promoted when inoculated with strain CS5.

Enrichment and isolation of a microbial culture capable of degrading

Endosulfan with minimal production of Endosulfan sulfate, the toxic metabolite of

Endosulfan, from tropical acid soil was reported by Surya Kalyani et al. (2009). This

was achieved by using the insecticide as sole sulfur source. In 20 days, enriched

microbial culture, SKL-1, later identified as Ps. aeruginosa, degraded up to 50.25 and

69.77% of α and β-Endosulfan respectively. By the 20th day of incubation, percentage

of bioformation of Endosulfan sulfate to total formation was 2.12%. Bacterial growth

reaching up to an optical density of 600 nm 2.34 and aryl sulfatase activity of the

60

broth reaching upto 23.93 μgpNP/ml/h was concomitant with degradation. It was

suggested by the results of this study that this novel strain was a valuable source of

potent Endosulfan-degrading enzymes for use in enzymatic bioremediation. Aryl

sulfatase activity of the broth increased with degradation of Endosulfan. This

suggested the probable involvement of the enzyme in the transformation of

Endosulfan to its metabolites.

Jayashree and Vasudevan (2007) studied the effect of Tween 80 added to the

soil on the degradation of Endosulfan by Pseudomonas aeruginosa at different pH

(7.0 and 8.5). The solubility and degradation of Endosulfan was enhanced by the

addition of synthetic surfactant Tween 80. Degradation of 94% was observed at pH

8.5 with Tween 80 added soil. The unit T4 inoculated at pH 8.5 showed 86% alpha

and 60% beta Endosulfan degradation, the bacterial population was 73x108 CFU/g of

soil. It was observed that both the isomers were degraded with formation of endodiol

and Endosulfan sulfate.

Endosulfan degradation by a mixed culture isolated from a pesticide-

contaminated soil was studied in batch experiments by Kumar et al. (2007). The

mixed culture was able to degrade 73% and 81% of α and β-Endosulfan respectively

after two weeks of incubation. Using GC/MS endodiol was identified as degradation

intermediate. Before and after degradation, the toxicity studies of Endosulfan were

carried out using micronucleus assay on human polymorphonuclear cells which

suggested that significant reduction in the toxicity was observed when Endosulfan

isomers were metabolized by the mixed culture. Studies were also carried out to

quantify the degradation potential of the individual species in the mixed bacterial

culture. Majority of the degradation by the mixed culture was carried out by the two

61

cultures which were identified as Stenotrophomonas maltophilia and Rhodococcus

erythropolis by 16S rRNA. Better degradation efficiency was shown by

S. maltophilia compared to that by R. erythropolis. This was the first report of

Endosulfan degradation using Stenotrophomonas maltophilia and Rhodococcus

erythropolis.

Kumar and Philip (2007) enriched a novel mixed bacterial culture from an

Endosulfan processing industrial surface soil. In both aerobic and anaerobic

conditions the cultures degraded aqueous phase Endosulfan. Endosulfan degradation

in silty gravel with sand (GM) was examined using the cultures via pilot scale reactor

at an Endosulfan concentration of 0.78+/-0.01 mg /g soil and optimized moisture

content of 40+/-1%. Vertical spatial variability in Endosulfan degradation was

observed during operation, within the reactor. At the end of 56 days, maximum

Endosulfan degradation efficiency of 78+/-0.2% and 86.91+/-0.2% was observed in

the top and bottom portion of the reactor, respectively. Within the reactor, both

aerobic and anaerobic conditions were observed. In anaerobic condition, Endosulfan

degradation was predominant and progressively down the soil depth, the total protein

concentration in the reactor declined. Throughout the study, known intermediate

metabolites of Endosulfan reported by previous researchers were not observed.

Through enrichment technique, Hussain et al. (2007b) isolated 29 bacterial

strains from 15 specific sites using Endosulfan as sole sulfur source. These bacterial

strains substantially differed in their potential to degrade Endosulfan in vitro ranging

from 40 to 93% of the spiked amount (100 mg/l). Within 14 days of incubation,

substantial decrease in pH of the broth from 8.2 to 3.7 was observed as a result of

biodegradation of Endosulfan by these bacteria. More than 90% of the spiked amount

62

(100 mg/l) of Endosulfan in the broth was consumed within 14 days of incubation.

The three bacterial strains, Ps. spinosa, Ps. aeruginosa and Burkholderia cepacia

were the most efficient degraders of both α- and β-Endosulfan. At an initial pH of 8.0

and at an incubation temperature of 30°C, maximum biodegradation by these three

selected efficient bacterial strains was observed which implied that these bacterial

strains could be employed for bioremediation of Endosulfan polluted soil and water

environments. It was revealed by High performance liquid chromatography analyses

that Endosulfan diol and Endosulfan ether were among the products of endosulfan

metabolism by these bacterial strains. A persistent and toxic metabolite of

Endosulfan, Endosulfan sulfate, was not detected in any case. GC-MS analysis further

confirmed the presence of Endosulfan diol and Endosulfan ether in the bacterial

metabolites.

Arshad et al. (2007) studied optimal environmental conditions for achieving

biodegradation of α and β-Endosulfan in soil slurries following inoculation with an

Endosulfan degrading strain of Ps. aeruginosa. Soil texture, soil slurry: water ratios,

initial inoculum size, pH, incubation temperature, aeration and the use of exogenous

sources of organic and amino acids were the parameters that were investigated. It was

shown by the results that Endosulfan degradation was most effectively achieved at an

initial inoculum size of 600 µl (OD = 0.86), incubation temperature of 30°C, in

aerated slurries at pH 8, in loam soil. More than 85% of spiked α and β-Endosulfan

(100 mg/l) was removed by the bacterium removed after 16 days under these

conditions. In different textured soils, biodegradation of Endosulfan varied being

more rapid in coarse textured soil than in fine textured soil. Less biodegradation of

Endosulfan was observed with increase in the soil contents in the slurry above 15%.

63

Exogenous application of organic acids and amino acids had stimulatory and

inhibitory effects, respectively, on biodegradation of Endosulfan.

From the contaminated soil of the premises of a pesticide manufacturing

industry, Kumar and Philip (2006a) isolated three novel bacterial species, namely,

Staphylococcus spp., Bacillus circulans-I, and Bacillus circulans-II. To assess their

potential for the degradation of aqueous Endosulfan in aerobic and facultative

anaerobic condition batch experiments were conducted using both mixed and pure

cultures. The effect of supplementary carbon (dextrose) source on Endosulfa n

degradation was also examined. In aerobic and facultative anaerobic conditions, with

an initial Endosulfan concentration of 50 mg/l mixed bacterial culture was able to

degrade 71.82+/-0.2% and 76.04+/-0.2% of Endosulfan respectively after four weeks

of incubation. Endosulfan degradation efficiency was amplified by 13.36+/-0.6% by

addition of dextrose in aerobic system and 12.33+/-0.6% in facultative anaerobic

system. To quantify the degradation potential of these individual species pure culture

studies were carried out. Staphylococcus spp. utilized more β-Endosulfan compared

to alpha Endosulfan in facultative anaerobic system, whereas Bacillus circulans-I and

Bacillus circulans-II utilized more alpha Endosulfan compared to β-Endosulfan in

aerobic system.

Kumar and Philip (2006b), obtained a mixed bacterial culture consisting of

Staphylococcus sp., Bacillus circulans-I and Bacillus circulans–II which were

enriched from contaminated soil collected from the vicinity of an Endosulfan

processing industry. Through batch experiments with an initial Endosulfan

concentration of 50 mg/l, the degradation of Endosulfan by mixed bacterial culture

was studied in aerobic and facultative anaerobic conditions. Mixed bacterial culture

64

was able to degrade 71.58+/-0.2% and 75.88+/-0.2% of Endosulfan in aerobic and

facultative anaerobic conditions, respectively after 3 weeks of incubation. In both the

conditions the addition of external carbon (dextrose) increased the Endosulfan

degradation. The optimal dextrose concentration and inoculum size was found to be 1

g/l and 75 mg/l, respectively. pH of the system had significantly affected Endosulfan

degradation. Compared to β-Endosulfan, the degradation of α-Endosulfan was more

in all the experiments. Using miniature and bench scale soil reactors Endosulfan

biodegradation in soil was evaluated. Endosulfan degradation efficiency in miniature

soil reactors were in the order of sandy soil followed by red soil, composted soil and

clayey soil in both aerobic and anaerobic conditions. Endosulfan degradation was

observed more in the bottom layers in bench scale soil reactors. Maximum

Endosulfan degradation efficiency of 95.48+/-0.17% was observed in red soil reactor

where as in composted soil-I (moisture 38+/-1%) and composted soil- II (moisture

45+/-1%) it was 96.03+/-0.23% and 94.84+/-0.19%, respectively after 4 weeks.

Endosulfan concentration in the leachate was increased by the high moisture content

in compost soil reactor-II.

Verma et al. (2006) isolated a Rhodococcus MTCC 6716 bacterial strain for

the first time from the gut microflora of an Indian earthworm (Metaphire posthuma).

92.58% Endosulfan was degraded within 15 days by the strain used as it used

Endosulfan as a source of carbon. The persistent form of the toxic metabolite

Endosulfan sulfate was not produced by the bacterium. Luxurious growth in minimal

medium with high concentrations of Endosulfan (80 mg/ml) was exhibited by this

strain. Nearly complete degradation of the insecticide was suggested as degradation of

Endosulfan occurred simultaneously with bacterial growth and an increase in chloride

65

ion (87.1%) in the growth medium. Degradation potential of the strain was retained

even under sunlight exposure. This strain was able to tolerate a temperature of 45°C.

From cotton-growing soil Shivaramaiah and Kennedy (2006) isolated a

bacterium capable of metabolizing Endosulfan which was effectively shown to

degrade Endosulfan into Endosulfan sulfate. Within 3 days of incubation, the

bacterium degraded 50% of the compound. Endosulfan sulfate was the only terminal

product and no other metabolites were formed during the incubation. Gas

chromatography was used to analyze Endosulfan and its metabolites. It was

concluded that the basis for the development of bioremediation strategies to remediate

the pollutants in the environment would be provided by his study.

Bacterium KS-2P was isolated by Lee et al. (2006) through repetitive

enrichment and successive subculture using Endosulfan or Endosulfan sulfate as the

sole carbon source. On the basis of the results of a 16S rDNA sequencing analysis

and MIDI test the KS-2P was identified as Pseudomonas spp. In minimal medium

containing Endosulfan (23.5 μg/ml) or Endosulfan sulfate (21 μg/ml), the degradation

ratios for Endosulfan or Endosulfan sulfate were 52% and 71%, respectively. It was

suggested by these results that Pseudomonas spp. KS-2P had potential as a biocatalyst

for Endosulfan bioremediation.

Kwon et al. (2005) isolated a bacterium capable of degrading Endosulfan

sulfate as well as Endosulfan through repetitive enrichment and successive subculture

using mineral salt medium, containing Endosulfan or Endosulfan sulfate as the sole

source of carbon and energy. From the results of 16S rDNA sequence analysis the

bacterium KE-8 was identified as Klebsiella oxytoca. The biomass was rapidly

increased to an optical density at 550 nm of 1.9 in 4 days in biodegradation assays

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with KE-8 using mineral salt medium containing Endosulfan (150 mg/l) or

Endosulfan sulfate (173 mg/l). It was further suggested that K. oxytoca KE-8 had

high potential as a biocatalyst for bioremediation of Endosulfan and/or Endosulfan

sulfate with the analysis of the metabolites.

Microorganisms, capable of degrading Endosulfan were enriched, isolated and

identified by Siddique et al. (2003). By using the insecticide as either the sole source

of carbon or sulfur in parallel studies, enrichment was achieved. Using Endosulfan as

a sole carbon source, bacteria (BF2 and B4) were selected. A Pandoraea spp. (Lin-3)

previously isolated in their laboratory using lindane (γ-HCH) as a carbon source was

also screened for Endosulfan degradation. Bacterial strains B4 and Lin-3 degraded α-

Endosulfan up to 79.6% and 81.8% and β-Endosulfan up to 83.9% and 86.8%,

respectively, in 15 days. Among the bacterial strains isolated by providing Endosulfan

as a sulfur source, B4s and F4t degraded α-Endosulfan by as much as 70.4% and

68.5% and β-Endosulfan by 70.4% and 70.8%, respectively, after 15 days.

Degradation of the insecticide occurred concomitant with bacterial growth reaching

an optical density (at 600 nm) of 0.366 and 0.322 for B4 and Lin-3, respectively.

With the other bacterial strains utilizing Endosulfan as a sulfur source also, high OD

(at 600 nm) was also noted. There was a significant decrease in the pH of the nutrient

culture media by these strains while growing on Endosulfan. It was observed that the

hydrolysis of Endosulfan resulted in the formation of an intermediate metabolite,

Endosulfan diol, which was further metabolized to Endosulfan ether.

Endosulfan degrading bacteria, which do not form toxic Endosulfan sulfate,

were isolated from various soil samples by Kwon et al. (2002) for bioremediation of

toxic Endosulfan, using Endosulfan as sole carbon and energy source. A total of 40

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bacteria were isolated. Among these, superior Endosulfan degradation activity was

shown by strain KE-1, which was identified as Klebsiella pneumoniae by

physiological and 16S rDNA sequence analysis. It was demonstrated by the analysis

of culture pH, growth, free sulfate and Endosulfan and its metabolites, that, KE-1

biologically degraded 8.72 μg Endosulfan/ml/d when incubated with 93.9 μg

Endosulfan/ml for 10 days without formation of toxic Endosulfan sulfate. It was

suggested by these results that K. pneumoniae KE-1 degraded Endosulfan by a non-

oxidative pathway and that strain KE-1 had the potential as a biocatalyst for

Endosulfan bioremediation.

From the soil with a history of Endosulfan exposure, Sutherland et al. (2000)

enriched an Endosulfan-degrading mixed bacterial culture. Enrichment was carried

out by using the insecticide as the sole source of sulfur. By using strongly buffered

culture medium (pH 6.6), chemical hydrolysis was minimized and to emulsify the

insecticide, the detergent Tween 80 was included, thereby increasing the amount of

Endosulfan in contact with the bacteria. Growth was not observed in control cultures

in the absence of Endosulfan. Degradation of the insecticide occurred concomitant

with bacterial growth with the oxidation and hydrolysis of the compound.

2.4. Degradation of Endosulfan by fungi

The possible nontoxic bioremediation of Endosulfan was investigated by

Kamei et al. (2011). From eight species of white-rot fungi, an Endosulfan degrading

fungus that does not produce Endosulfan sulfate was selected. In cultures of Trametes

hirsuta high degradation of Endosulfan and low accumulation of Endosulfan sulfate

were found. A degradation experiment using Endosulfan sulfate as the substrate

revealed that T. hirsuta was able to further degrade Endosulfan sulfate following the

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oxidative conversion of Endosulfan to Endosulfan sulfate. Through hydrolytic

pathways, Endosulfan and Endosulfan sulfate were converted to several metabolites.

In addition, in the T. hirsuta culture containing Endosulfan sulfate, Endosulfan

dimethylene, previously reported as a metabolite of the soil bacterium Arthrobacter

sp., was detected. It was suggested by these results that as T. hirsuta had multiple

pathways for the degradation of Endosulfan and Endosulfan sulfate, it had great

potential for use as a biocatalyst in Endosulfan bioremediation.

Goswami et al. (2009) studied the biodegradation of Endosulfan and the

metabolites formed using fungi both in broth culture as well as in soil microcosm.

After isolation of fungal strains from soil, they were grown in broth Czapek-dox

medium. For detailed studies, the strain which utilized Endosulfan and showed

maximum growth was selected. Aspergillus sydoni showed maximum degrading

capability in shake flask culture which degraded 95% of Endosulfan α and 97% o f

Endosulfan β in 18 days of incubation. In six different treatments, soil microcosm

study was also carried out using this strain. It was concluded that this isolated fungal

strain would be a potential source for Endosulfan degrading enzymes and could be

used for bioremediation at the contaminated sites.

Bhalerao and Puranik (2007) studied the enrichment, isolation and screening

of fungi capable of metabolizing Endosulfan. A total of 16 fungal isolates were

obtained by enrichment from soil samples exposed to Endosulfan. Screening of the

isolates was done using a gradient plate assay and by broth assay the results were

confirmed. For further studies, an isolate identified as Aspergillus niger was selected

on the basis of tolerance to Endosulfan. The culture tolerated 400 mg/ml of technical

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grade Endosulfan. After 12 days of incubation, complete disappearance of Endosulfan

was seen.

From contaminated soils efficient Endosulfan-degrading fungal strains were

isolated by Husssain et al. (2007a). From fifteen specific sites, sixteen fungal strains

were isolated. This was done by employing enrichment techniques while using

Endosulfan as a sole sulfur source. These strains were tested for their potential to

degrade Endosulfan. It was observed that both α and β-Endosulfan were degraded

upto 75% by Chaetosartorya stromatoides, Aspergillus terricola, and Aspergillus

terreus in addition to ~20% abiotic degradation of the spiked amount (100 mg/l) in

the broth within 12 days of incubation. A substantial decrease in pH of the broth from

7.0 to 3.2 was seen with biodegradation of Endosulfan by soil fungi. At an initial

broth pH of 6, incubation temperature of 30°C and under agitation conditions

maximum biodegradation of Endosulfan by these selected fungal strains was found.

It was indicated by this study that the isolated strains carried efficient enzyme systems

required for bioremediation of Endosulfan-contaminated soil and water environments.

Isolation and identification of two strains of fungi (F1 and F4) capable of

degrading Endosulfan was done by Siddique et al. (2003). By using the insecticide as

either the sole source of carbon or sulfur in parallel studies, enrichment was achieved.

Within 15 days of incubation F1and F4 (Fusarium ventricosum) degraded α-

Endosulfan by as much as 82.2% and 91.1% and β-Endosulfan by 78.5% and 89.9%,

respectively.

2.5. Degradation pathway of Chlorpyrifos

Anwar et al. (2009), Xu et al. (2008), Yang et al. (2006) and Singh et al.

(2004) suggested that in most cases, the degrading microorganisms tend to metabolize

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Chlorpyrifos by hydrolysis to form diethylthiophosphoric acid (DETP) and TCP.

According to Theriot and Grunden (2011) and Sogorb and Vilanova (2002), it was

generally considered that ester hydrolysis was the primary degradation pathway of

Chlorpyrifos. However, Xu et al. (2008) suggested that due to its resistance to

enhanced degradation, studies on further metabolism and identification of

intermediate products of Chlorpyrifos have not been extensive. TCP has been found

to be the major metabolite of Chlorpyrifos degradation, though other metabolites have

been reported by a few investigators. During the degradation of Chlorpyrifos by

P. aeruginosa, Vidya Lakshmi et al. (2009) observed the formation of 3, 5, 6-

trichloro-2-pyridinol, the major metabolite of Chlorpyrifos degradation, which

disappeared to negligible amounts. Li et al. (2007b) studied a highly effective

Chlorpyrifos-degrading bacterium strain Dsp-2, which utilized Chlorpyrifos as its sole

source of carbon for growth, by hydrolyzing Chlorpyrifos to 3, 5, 6-trichloro-2-

pyridinol (TCP). Sasikala et al. (2012) showed the presence of metabolites

chlopyrifos-oxon and Diethylphosphorothioate during Chlorpyrifos degradation using

bacterial cultures by LC-mass spectral analysis.

Pathways for Chlorpyrifos degradation by microorganisms have been studied

by various scientists. Chen et al. (2012) observed that degradation of Chlorpyrifos

was accompanied by a transient accumulation of TCP. Moreover, it was observed that

TCP was the only intermediate product that disappeared quickly and was further

transformed without any other persistent metabolites by strain Hu-01. Finally, after 6

days of incubation no persistent accumulative metabolite was detected by GC-MS. It

was suggested that some other metabolites might have formed and been immediately

degraded by the fungus. Gao et al. (2012) purified Chlorpyrifos hydrolase from the

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fungus. The metabolic pathway of Chlorpyrifos was proposed based on these results

of Chen et al. (2012) and Gao et al. (2012).

Studies on further metabolism and identification of intermediate products of

the phosphorus containing products have not been extensive. Feng et al. (1997)

isolated a Pseudomonas spp. which can mineralize TCP in liquid medium. Later the

same group, on the basis of combined experiments with photolysis and microbial

degradation, suggested that TCP was metabolized by a Pseudomonas spp. by a

reductive dechlorination pathway (Feng et al., 1998). In this pathway, TCP is first

reductively dechlorinated into chlorodihydro-2-pyridone, which is further

dechlorinated to tetra-hydro-2-pyridone. Ring cleavage of this compound resulted in

formation of maleamide semialdehyde, which is metabolized to water, carbon dioxide,

and ammonium ions. The postulated pathway steps include hydrolysis, yielding

monoester and finally inorganic phosphate (Fig. 5) (Singh and Walker, 2006).

2.6. Degradation pathway of Endosulfan

Commercially available Endosulfan exists as two diastereoisomers, α and β, in

a ratio of 7:3. The former is more volatile and thought to be more toxic (Siddique et

al., 2003; Lee et al., 2006). Some microbial enzymes are specific to one isomer or

catalyze at different rates for each isomer (Kwon et al., 2005).

For example, a Mycobacterium tuberculosis ESD enzyme degrades β-

Endosulfan to the monoaldehyde and hydroxyether (depending on the reducing

equivalent stoichiometry), but transforms α-Endosulfan to the more toxic Endosulfan

sulfate (Sutherland et al., 2002b). However, oxidation of Endosulfan or Endosulfan

sulfate by the monooxygenase encoded by ese in Arthrobacter sp. KW yields

Endosulfan monoalcohol (Weir et al., 2006).

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Fig. 5: Degradation pathway of Chlorpyrifos (Singh and Walker, 2006)

A bacterium capable of metabolizing Endosulfan was isolated by

Shivaramaiah and Kennedy (2006), from cotton-growing soil and was effectively

shown to degrade Endosulfan into Endosulfan sulfate. The metabolites formed

indicated that the organism follows an oxidative pathway for metabolism of this

pesticide. Endosulfan and its metabolites were analyzed by gas chromatography and

it was found that Endosulfan sulfate was the only terminal product and no other

metabolites were formed during the incubation.

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Miles and May (1979), proposed a pathway wherein Endosulfan is converted

to Endosulfan sulfate followed by Endosulfan diol, Endosulfan hydroxyether and

Endosulfan lactone (Fig. 6a). Formation of these metabolites has also been confirmed

by Katayma and Matsumura (1993) and Kullman and Matsumura (1996) while

working with fungi.

Fig. 6a: Degradation pathway of Endosulfan (Miles and May, 1979)

Sutherland et al. (2000) reported that Endosulfan was both oxidized and

hydrolyzed. The oxidation reaction favored the alpha isomer and terminal pathway

product, endosulfate, was produced. Hydrolysis involved a novel intermediate, which

on the basis of gas chromatography-mass spectrometry and chemical derivatization

results, was tentatively identified as Endosulfan monoaldehyde. It was suggested by

the accumulation and decline of metabolites that the parent compound was hydrolyzed

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to the putative monoaldehyde, thereby releasing the sulfite moiety required for

growth. The monoaldehyde was then oxidized to Endosulfan hydroxyether and further

metabolized to polar product(s). Oxidation of Endosulfan or the formation of other

metabolites was not prevented by the cytochrome P450 inhibitor, piperonyl butoxide

(Fig. 6b).

Fig. 6b: Degradation pathway of Endosulfan (Sutherland et al., 2000)

The possible nontoxic bioremediation of Endosulfan was also investigated by

Kamei et al. (2011). Further degradation of Endosulfan sulfate following the

oxidative conversion of Endosulfan to Endosulfan sulfate by T. Hirsuta was revealed

by a degradation experiment using Endosulfan sulfate as the substrate. Endosulfan

and Endosulfan sulfate were converted to several metabolites via hydrolytic pathways.

In addition, Endosulfan dimethylene, previously reported as a metabolite of the soil

bacterium Arthrobacter sp., was detected in T. hirsuta culture containing Endosulfan

sulfate. These results suggested that T. hirsuta had multiple pathways for the

75

degradation of Endosulfan and Endosulfan sulfate and thus has great potential for use

as a biocatalyst in Endosulfan bioremediation.

Alternatively, hydrolysis of Endosulfan in some bacteria (Ps. aeruginosa,

Burkholderia cepaeia) yields the less toxic metabolite Endosulfan diol (Kumar et al.,

2007). Endosulfan can spontanteously hydrolyze to the diol in alkaline conditions, so

it is difficult to separate bacterial from abiotic hydrolysis (Sutherland et al., 2002b).

The diol can be converted to Endosulfan ether (Hussein et al., 2007b) or Endosulfan

hydroxyether (Lee et al., 2003) and then Endosulfan lactone (Awasthi et al., 2003).

Hydrolysis of Endosulfan lactone yields Endosulfan hydroxycarboxylate (Walse et

al., 2003). These various branches of Endosulfan degradation all result in

desulfurization while leaving the chlorines intact, exhibiting the recalcitrance to

bioremediation found in many organohalogen aromatics

(http://umbbd.ethz.ch/end/end_map.html).

Degradation of Endosulfan can also occur anaerobically. Batch experiments

using bacterial consortium and its pure isolates for their potential degradation of

Endosulfan and its metabolites, i.e., Endosulfan sulfate, Endosulfan ether and

Endosulfan lactone, in anaerobic condition were conducted by Kumar and Philip,

2006c. It was observed that Endosulfan degradation was promising with bacterial

consortium and pure isolates. β-Endosulfan was preferably utilized by Staphylococcus

spp. whereas more of α-Endosulfan was utilized by the other two Bacillus strains.

Degradation of Endosulfan ether and Endosulfan lactone was promising with Bacillus

circulans I and II whereas no Endosulfan sulfate was degraded by any of these strains.

This investigation postulated that Endosulfan was mineralized via hydrolysis pathway

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with the formation of carbenium ions and/or ethylcarboxylates, which later converted

into simple hydrocarbons.

2.7. Studies on the efficiency of immobilized bacteria in pesticide degradation

Research has suggested that higher productivity results from cellular or

genetic modifications induced by immobilization. Evidences indicating that the

immobilized cells are much more tolerant to perturbations in the reaction environment

and less susceptible to toxic substances make immobilized cell systems particularly

attractive for treatment of toxic substances like pesticides (Kim et al., 2002; Jianlong

et al., 2002; Manohar et al., 2001). Literatures are available on use of immobilized

cells and enzymes for the degradation of organophosphate and organochlorine

pesticides.

Methyl parathion degradation was optimized by Abdel-Razek et al. (2013),

using a strain of Escherichia coli DH5α expressing the opd gene. It was indicated by

the results that this strain had lower enzymatic activity compared to the

Flavobacterium sp. ATCC 27551 strain from which the opd gene was derived. Both

strains were assessed for their ability to degrade Methyl parathion (MP) in a mineral

salt medium with or without the addition of glucose either as suspended cells or

immobilized on tezontle, a volcanic rock. Both strains degraded Methyl parathion

with similar efficiencies, but immobilized cells degraded Methyl parathion more

efficiently than cells in suspension.

For biological treatment of Methyl-parathion and Tetrachlorvinphos, Yañez-

Ocampo et al. (2011) evaluated a tezontle-packed up-flow reactor (TPUFR) with an

immobilized bacterial consortium. Four flow rates (0.936, 1.41, 2.19 and 3.51 l/h)

and four hydraulic residence times (0.313, 0.206, 0.133 and 0.083 h) were evaluated

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in a TPUFR with the aim of developing a tool for pesticide biodegradation. In the

bioreactor, 75% efficiency in the removal of methyl-parathion and tetrachlorvinphos

was obtained with an operating time of eight hours and a flow of 0.936 l/h. Their

adsorptions in the volcanic rock were 9% and 6% respectively. It was demonstrated

that the biological activity of the immobilized bacterial consortium led to the removal

of pesticides. The decrease in toxicity in the treated effluent from the bioreactor was

confirmed through the application of acute toxicity tests on Eisenia foetida. It was

concluded that immobilization of a bacterial consortium using tezontle as a support

was innovative and an economical tool for the treatment of mixtures of

organophosphorus pesticide residues.

Jo et al. (2010) examined the Endosulfan degrading ability of Klebsiella

oxytoca KE-8 immobilized by entrapment with activated carbon. With the

immobilized bacterial strains on several different activated carbon based support

materials, Endosulfan degradation was investigated. Activated carbon (8×30 mesh)

was chosen as a support material based on results. In batch shake flask cultures, the

immobilized Klebsiella oxytoca KE-8, with the cell density of 4 mg/g (dry weight)

degraded 22.18 µg /ml Endosulfan within 5 days at pH 7.0 and 30°C. For Endosulfan

degradation, experimentation with recycle packed bed column mode and continuous

packed bed column mode was also done. The immobilized cells in a laboratory scale

pack bed column with support beads were able to degrade Endosulfan completely in

defined minimal salt medium at a maximum rate of 129.6 µg/ml per day under

optimum operation condition. Without significant decrease, the Endosulfan

degradation activity could be demonstrated at 4 °C for one month. It was suggested by

the results of this study that immobilized cells of Klebsiella oxytoca KE-8 might be

applicable to Endosulfan contaminated site.

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Effective degradation of Chlorpyrifos was demonstrated by Xie et al. (2010)

using the free enzyme extracted from WZ-I, which was identified as Fusarium LK. ex

Fx. The properties of the immobilized enzyme were compared with those of the free

enzyme. In a solution of 30 g/l sodium alginate at 4°C for 4–12 h, the optimal

immobilization of the enzyme was achieved. At pH 8.0 and 45°C the immobilized

enzyme showed the maximal activity. Compared to that of the free enzyme, the

maximum initial rate and the substrate concentration of the immobilized enzyme were

less. Therefore, the immobilized enzyme had a higher capacity to withstand a broader

range of temperatures and pH conditions than the free enzyme. The immobilized

enzyme was more active than the free enzyme in the degradation reaction with

varying pH and temperatures. Even after three repeated uses, the immobilized enzyme

exhibited only a slight loss in its initial activity. It was shown by the results that the

immobilized enzyme was more resistant to different environmental conditions,

suggesting that it was viable for future practical use.

In order to remove Methyl-parathion (MP) and Tetrachlorvinphos (TCF),

Yañez-Ocampo et al. (2009) immobilized a bacterial consortium with two supports

consisting of alginate beads or stones of tezontle colonized by biofilm. For suspended

and immobilized consortium using a mineral salt medium supplemented with MP and

TCF at 25 mg/l and with 0.1% (w/v) glucose as a co-substrate, removal kinetics was

recorded. For the consortium cultivated in suspension the viability was maintained for

6 days, whereas the viability of the consortium immobilized in alginate and tezontle

supports was maintained for up to 11 and 13 days, respectively. When glucose was

used as a co-substrate, growth was enhanced. When consortium was immobilized in

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alginate beads and biofilm on tezontle the percentage of MP removed was

significantly higher (α = 0.05) as compared to suspension culture.

For the detoxification and biodegradation of Coumaphos, an organophosphate

insecticide and its hydrolysis products, chlorferon and diethlythiophosphate (DETP),

Ca-alginate immobilized cell systems were developed by Ha et al. (2009). For

bioreactor operation, 200 g beads/l and 300 g beads/l was the optimum bead loading

for chlorferon degradation and for DETP degradation respectively. The degradation

rate for an immobilized consortium of chlorferon-degrading bacteria was five times

greater than that for freely suspended cells and hydrolysis of Coumaphos by

immobilized OPH+ Escherichia coli was 2.5 times greater when using waste Cattle

Dip (UCD) solution was used as substrate. The degradation by immobilized cells was

enhanced primarily due to protection of the cells from inhibitory substances present in

the UCD solution. Increased reaction rates would also be due to physiological

changes of the cells caused by Ca-alginate immobilization. It was observed that

degradation rates for repeated operations increased for successive batches which

indicated that cells became better adapted to the reaction conditions over time.

Mansee et al. (2005) developed a cost-effective method for the production and

immobilization of Organophosphate hydrolase (OPH) in a pilot application in an

enzyme bioreactor column for detoxification of Paraoxon and Coumaphos in

contaminated wastewaters. For allowing one-step purification and immobilization of

OPH on cheap and abundantly available cellulose immobilization matrices, a fusion

between OPH and a cellulose binding domain that binds selectively to cellulose was

generated. The immobilized fusion enzyme was able to completely degrade

Coumaphos up to a concentration of 0.2 mM when packed in a column bioreactor.

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Stirring of OPH immobilized on cellulose materials resulted in complete

organophosphate degradation of 1.5 mM Coumaphos. For about 2 months, the

bioreactor column degraded the compounds tested at high concentration, rapidly and

without loss of process productivity.

By enrichment of soil samples collected from paddy fields in Japan

Pattanasupong et al. (2004) obtained a bacterial consortium capable of simultaneously

degrading the fungicide, Carbendazim and the herbicide, 2, 4- dichlorophenoxyacetic

acid (2,4-D). This consortium was acclimated in a continuously fed culture with 20

µM Carbendazim and 2 mM 2, 4-D as sole carbon sources using a glass column

reactor. Up to 100 µM Carbendazim and 3 mM 2, 4-D was completely degradaed by

this acclimated consortium within 36 and 24 hours, respectively, in batch culture. A

lag time was observed after precultivation in a rich medium. Compared with the use

of free cells, the degradation ability of this consortium was enhanced with the

immobilization of the consortium on a polyester support. It was suggested that this

microbial consortium could be useful for bioremediation at sites contaminated with

these pesticides.

Qiao et al. (2003) studied biodegradation of pesticides using immobilized

recombinant Escherichia coli.

Kim et al. (2002) cultivated a recombinant strain of Escherichia coli in a rich

medium containing all essential nutrients. This strain contained the opd gene for

organophosphate hydrolase (OPH), which is capable of active hydrolysis of

organophosphate neurotoxins including Chlorpyrifos. Cells were harvested and

utilized in lab scale experiments. These were used in the form of either freely

suspended cells or cells immobilized within a macroporous gel matrix, poly (vinyl

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alcohol) (PVA) cryogel. Compared to rates with the microbial consortium naturally

present in Cattle Dip Waste, significantly higher degradation rates were achieved with

either suspended or immobilized OPH+ cells. Of the two nongrowing cell systems,

the detoxification rate with immobilized cells was approximately twice that of freely

suspended cells. Kinetic studies demonstrated that a higher and maximum reaction

rate was achieved with the immobilized cell system. Over a 4 month period of use and

storage, the immobilized cells retained their activity demonstrating both sustained

catalytic activity and long-term mechanical stability.

Immobilization of recombinant E. coli cells with OPH on the cell surface in

highly porous sintered glass beads and the subsequent application of the immobilized

cell in a continuous-flow packed bed bioreactor for the biodetoxification of a widely

used insecticide, Coumaphos were reported by Mansee et al. (2000).

Richins et al. (2000) showed that bifunctional fusion proteins consisting of

organophosphate hydrolase (OPH) moieties linked to a Clostridium-derived cellulose-

binding domain (CBD) were highly effective in degrading organophosphate nerve

agents, enabling purification and immobilization onto different cellulose materials in

essentially a single step. Using paraoxon as the substrate, enzyme kinetics studies

were performed for the CBD–OPH fusions. The kinetics values of the unbound

fusion enzymes were similar to OPH with a modest increase in Km. A further increase

in the Km values of approximately twofold was recorded as a result of immobilization

of the enzymes onto microcrystalline cellulose. There was only a minimal effect seen

on the pH profile of the cellulose immobilized enzymes. The CBD–OPH fusion

proteins could be immobilized onto a variety of cellulose matrices with the retention

of up to 85% of their original activity for 30 days. In an immobilized enzyme reactor

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repeated hydrolysis of paraoxon was achieved with 100% degradation e fficiency over

45 days. Thus it was suggested that these fusion proteins would prove to be invaluable

tools for the development of low cost, OPH-based cellulose materials for the

simultaneous adsorption and degradation of stored or spilled organophosphate wastes.

An improved whole-cell technology for detoxifying organophosphate nerve

agents was developed by Mulchandani et al. (1999) based on genetically engineered

Escherichia coli with organophosphorus hydrolase anchored on the surface. The

immobilization of these novel biocatalysts on nonwoven polypropylene fabric and

their applications in detoxifying contaminated wastewaters was reported by them. The

immobilized cells degraded 100% of 0.8 mM paraoxon, a model organophosphate

nerve agent, in approximately 100 min, at a specific rate of 0.160 mM/min/g cell dry

wt, in batch operations. The immobilized cells retained almost 100% activity during

the initial six repeated cycles. Close to 90% activity was retained even after 12

repeated cycles, extending over a period of 19 days without any nutrient

supplementation. Other commonly used organophosphates, such as Diazinon,

Coumaphos, and Methylparathion were hydrolyzed efficiently in addition to

Paraoxon.

Both native and recombinant OPH’s have been immobilized onto nylon

(membrane, powder and tubing), porous glass and silica beads and used as enzyme

reactors for the detoxification of organophosphates (Caldwell and Raushel, 1991a;

1991b; Munnecke, 1979).

OPH has also been immobilized within polyurethane foams that can be

applied as sponges or wipes for the clean-up of pesticide spills (LeJuene and Russell,

1996; Havens and Rase, 1993).

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Mac Rae (1985) used immobilized cells to degrade organochlorine pesticides

like Lindane. They studied the removal of Lindane (γ- isomer of 1,2,3,4,5,6-

hexachlorocyclohexane), 2.4-D (2,4-dichlorophenoxyacetic acid) and 2,4,5-T (2,4,5-

tichlorophenoxyacetic acid) from water by microbial cells immobilized on magnetite.

With the use of cells of a yeast, two bacteria and an alga the removal of Lindane

ranged from 29 to 57%. It was reported that a bacterium, Rhodopseudomonas

sphaeroides had the highest sorption factor and no affect of pH in the range pH 4.0–

8.0 on sorption of Lindane by this bacterium was found. After 1 min sorption of

Lindane by R. sphaeroides reached equilibrium. There was no significant difference

between sorption of Lindane by live cells and sorption by heat killed cells.

Employing fresh cells of R. sphaeroides on magnetite, 90% of the Lindane from a

water sample was removed by four sorption stages. In the four stage process

magnetite alone removed 70% of the Lindane. During a mixing period of 1 h, cells of

Alcaligenes eutrophus removed 81%, 2, 4-D; 21.4% Lindane and 12.6% of the 2, 4, 5-

T added to water samples. By a mixture of A. eutroplus and R. sphaeroides cells

immobilized on magnetite 76.4%, 2, 4-D and 33% lindane from water samples

containing the two pesticides was removed.

2.8. Enzymes involved in organophosphate and organochlorine degradation

Microbes, their catabolic gene and respective enzymes have been isolated and

identified by several workers capable of degrading the pesticides. Genetic studies of

microbial degradation indicate that the plasmids are the main place for the gene of

interest usually spread throughout the microbial community. After, understanding the

gene of interest and enzyme involved, the Superbugs can be created to achieve the

desired result at fast rate in short time frame (Singh, 2008).

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Several enzymes capable of detoxifying organophosphates have been isolated

from microorganisms able to use organophosphates as a carbon source. The most

widely characterized phosphotriesterase is the bacterial organophosphate hydrolase

(OPH, E.C.8.1.3.1), which has been isolated from both Flavobacterium sp. ATCC

27551(Mulbry and Karns, 1989b) and Pseudomonas diminuta MG (Serdar et al.,

1989). It is one of the most crucial enzymes in the detoxification of organophosphorus

compounds, such as Paraoxon, Parathion, Coumphos and Diazion (Wu et al., 2002).

However, the enzyme does not catalyze the cleavage of carbonyl groups such as those

found in p-nitrophenyl acetate (Raushel, 2002).

Thengodkar and Sivakami (2010) observed that cultures of Spirulina platensis

grew in media containing up to 80 ppm of Chlorpyrifos due to an alkaline

phosphatase (ALP) activity that was detected in cell free extracts of Spirulina

platensis. Using ammonium sulfate precipitation and gel filtration this activity was

purified from the cell free extracts and was shown to belong to the class of EC 3.1.3.1

ALP. The purified enzyme degraded 100 ppm Chlorpyrifos to 20 ppm in 1 h,

transformed it into its primary metabolite 3, 5, 6-trichloro-2-pyridinol.

Based on the formation of clear haloes on Luria-Bertani plates overlaid with

Chlorpyrifos, Cho et al. (2004) used five improved variants which were selected from

two rounds of directed evolution. One variant, B3561, exhibited a 725-fold increase

in the kcat/Km value for Chlorpyrifos hydrolysis as well as enhanced hydrolysis rates

for several other OP compounds tested. Considering that wild-type OPH hydrolyzes

Paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in

hydrolysis of Paraoxon by B3561 suggested that this variant was one of the most

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efficient enzymes available to attack a wide spectrum of organophosphate nerve

agents.

Xie et al. (2005) studied the degradation characteristics of Chlorpyrifos

insecticides which were determined by the crude enzyme extracted from the isolated

strain WZ-I (Fusarium LK. ex Fx). They studied the best separating condition and

the degrading characteristic of Chlorpyrifos. Using its intracellular enzyme,

extracellular enzyme and cell fragment, rate of degradation for Chlorpyrifos was

60.8%, 11.3% and 48%, respectively. The degrading enzyme was extracted after this

fungus was incubated for 8 generations in the condition of non inducement and its

enzymic activity lost less, the results show that this enzyme is an intracellular and

connatural enzyme. The solubility protein of the crude enzyme was determined with

Albumin (bovine serum) as standard protein and the solubility protein of the crude

enzyme was 3.36 mg/ml. The pH optimum for crude enzyme was 6.8 for enzymatic

degradation of Chlorpyrifos, and it had comparatively high activity in the range of pH

6.0 - 9.0. The optimum temperature for enzymatic activity was at 40°C, it still had

comparatively high activity in the range of temperature 20-50°C, the activity of

enzyme rapidly reduced at 55°C, its activity was 41% of the maximal activity. The

crude enzyme showed Km value for Chlorpyrifos of 1.049 26 mM/l, and the maximal

enzymatic degradation rate was 0.253 5 µM/mg/min. Additional experimental

evidence suggests that the enzyme had the stability of endure for temperature and pH,

the crude enzyme of fungus WZ-I could effectively degrade Chlorpyrifos.

The bacterium Enterobacter strain B-14, isolated by Singh et al. (2004), had

very strong phosphotriesterase (OPH) activity and hydrolyzed 35 mg/l concentration

of Chlorpyrifos within 24 h when inoculated with 106 cells/g of soil.

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Cho et al. (2002) used sequential cycles of DNA shuffling and screening to

fine-tune and enhance the activity of OPH towards poorly degraded substrates. OPH

variants were displayed on the surface of Escherichia coli using the truncated ice

nucleation protein because of the inaccessibility of these pesticides across the cell

membrane. This was done in order to isolate novel enzymes with truly improved

substrate specificities. A solid-phase top agar method based on the detection of the

yellow product p-nitrophenol was developed for the rapid pre-screening of potential

variants with improved hydrolysis of methyl parathion. Several improved variants

were isolated after carrying out two rounds of DNA shuffling and screening. Methyl

parathion was hydrolyzed 25-fold faster than the wild type by one variant in

particular, 22A11.

Chen and Mulchandani (1998) showed that cultures with surface-expressed

OPH degraded Parathion and Paraoxon very effectively without the diffusional

limitation observed in cells expressing OPH intracellularly and also exhibited a very

long shelf- life, retaining 100% activity over a period of one month.

Rowland et al. (1991) worked on a heterologous phosphotriesterase (parathion

hydrolase), previously cloned from a Flavobacterium species into Streptomyces

lividans which was secreted at high levels and purified to homogeneity. It was

revealed by N-terminal analysis that it had been processed in the same manner as the

native membrane-bound Flavobacterium hydrolase. As determined by sodium

dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme consisted of a single

polypeptide with an apparent molecular weight of 35,000 Da. The characteristics of

the purified recombinant Parathion hydrolase were the same as the native

Flavobacterium hydrolase. A source of milligram quantities of Parathion hydrolase

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for future structural and mechanism studies was provided by this system and had the

potential to be used in toxic waste treatment strategies.

Three unique parathion hydrolases were purified from gram-negative bacterial

isolates and characterized by Mulbry and Karns (1989a). All three purified enzymes

had roughly comparable affinities for ethyl parathion and had broad temperature

optima at ca. 40°C.

Karns et al. (1987) used the hydrolase- producing Flavobacterium strain in a

pilot-scale system to detoxify waste containing high concentrations of the

organophosphate insecticide Coumaphos.

Aerobic bacteria degrading Endosulfan were isolated from contaminated

sludge by Yu et al. (2012). LD-6 was one of the isolates, which was identified as

Stenotrophomonas sp. Endosulfan as the sole source of carbon and sulfur could be

utilized by the bacterium. Within 10 days, 100 mg/l of Endosulfan was completely

degraded. Endosulfan diol and Endosulfan ether were detected as major metabolites

with a slight decrease in culture pH. It was indicated by the results that

Stenotrophomonas. sp. LD-6 might degrade Endosulfan by a non-oxidative pathway.

Biodegradation of both isomers was relatively better at a temperature range of 25–

35°C, with a maximum at 30°C. Cell crude extract of strain LD-6 could metabolize

Endosulfan rapidly and degradative enzymes were intracellular distributed and

constitutively expressed. Besides, application of the strain was found to promote the

removal of Endosulfan in soil.

Shivaramaiah and Kennedy (2006) studied biodegradation of Endosulfan by a

soil bacterium S3 which consistently degraded Endosulfan. Endosulfan degradation

results indicated that the enzyme system responsible was probably a mono-oxygenase,

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converting Endosulfan to Endosulfan sulfate. Both substrate disappearance and

product formation demonstrated the bacterial metabolism of Endosulfan.

Katayama and Matsumura (1993) showed that the cultures of Trichoderma

harzianum were capable of producing Endosulfan diol as a principal metabolite. They

suggested that a hydrolytic enzyme sulfatase is responsible for the indirect formation

of Endosulfan diol by hydrolysis of Endosulfan sulfate.

2.9. Genes involved in degradation of Chlorpyrifos

Various organophosphate-degrading genes (opd genes) have been isolated

from different species, some of which could degrade Chlorpyrifos (Horne et al., 2002;

Richnis et al., 1997; Serdar et al., 1982). The molecular basis of degradation of

certain organophosphates has been studied extensively (Horne et al., 2002; Mulbry et

al., 1986 and Serdar, 1982).

Detailed studies have focused on the activities of two bacterial isolates,

Flavobacterium spp. strain ATCC 27551 and Pseudomonas diminuta MG (Brown,

1980; Munnecke, 1976 and Sethunathan and Yoshida, 1973). Although the reported

hydrolase activities of these two strains differed in several respects, the genes

responsible (termed opd genes for organophosphate degradation) have been cloned

from both organisms and shown to be closely related by southern hybridization

(Mulbry et al., 1987; Mulbry et al., 1986; Serdar and Gibson, 1985). A bacterial

isolate possessing parathion hydrolase activity has been shown by Southern

hybridization to contain DNA homologous to the Flavobacterium opd gene

(Chaudhry et al., 1988).

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Singh et al. (2004) isolated an Enterobacter sp. from a soil from Australia

which showed enhanced degradation of Chlorpyrifos. It was suggested by the studies

of the molecular basis of degradation that the degrading ability could be polygenic

and chromosome based. It was revealed by further studies that the strain possessed a

novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a

different sequence from the widely studied organophosphate-degrading gene (opd).

Although the initial step in Chlorpyrifos degradation by the Enterobacter sp. was

similar to other OPH activities, the genes encoding the enzymes were different. It

was seen that amplification of isolated Enterobacter sp. DNA with three different sets

of primers designed from the conserved region of opd did not give any PCR products.

Hybridization of isolate DNA with the opd probe also failed, suggesting that the DNA

sequence of the gene responsible for Chlorpyrifos degradation is not similar to that of

the known opd gene. In the bacterium isolated in the present studies, no p lasmid

DNA was detected. This suggested that the Chlorpyrifos-degrading gene may be

chromosome based.

A widely distributed organophosphate-degrading gene (opd) was isolated from

temporally, geographically and biologically different species (Horne et al., 2002;

Serdar et al., 1982; Sethunathan and Yoshida, 1973). Most of the opd genes are

plasmid based (Mulbry et al., 1986). However, Horne et al., (2002) isolated an opd

gene from Agrobacterium radiobacter, which was located on the chromosome but had

a similar sequence to the opd gene from other bacteria. There have been reports of

organophosphate degradation genes with similar function but different sequences

from the opd gene: for example, a Methyl parathion-degrading Plesiomonas species

had a DNA sequence quite different from those of the known opd genes (Zhongli et

al., 2001). Coumaphos degrading Nocardioides simplex NRRL B-24074 (Mulbry,

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2000) has been reported to have novel organophosphate-degrading enzyme and gene

systems.

A bacterial strain, Cupriavidus sp. DT-1, capable of degrading Chlorpyrifos

and 3, 5, 6-trichloro-2-pyridinol (TCP) and using these compounds as sole carbon

source was isolated and characterized by Lu et al. (2013). The mpd gene, encoding

the enzyme responsible for Chlorpyrifos hydrolysis to TCP, was cloned and expressed

in Escherichia coli BL21.

A Chlorpyrifos degrading bacterium Ps. putida MAS-1 was isolated from the

cotton grown soil of NIAB, Faisalabad, Pakistan by Ajaz et al. (2009). Genetic

studies based on plasmid curing and electroporation mediated transformation were

performed on this bacterium. The bacterium lost the property to grow on the nutrient

agar containing 10 mg/ml Chlorpyrifos after acridine orange mediated curing. The

plasmid (bearing Chlorpyrifos degrading determinants/genes) was isolated and

transferred into E. coli DH5α. The transformants however, could not resist and grow

in the Chlorpyrifos containing medium. It may be concluded that Chlorpyrifos

degradation Ps. putida MAS-1 is accomplished by the combined action of plasmid

and chromosomal genes.

The gene encoding the Chlorpyrifos hydrolytic enzyme from a strain Dsp-2

was cloned by PCR by Li et al. (2007b). Although BLAST sequence search results

indicated that this gene has 99% similarity to mpd (a gene encoding the parathion-

methyl hydrolyzing enzyme in Plesiomonas sp. M6), its hydrolytic efficiency for

Chlorpyrifos was significantly greater than the wild-type mpd from strain M6.

Yang et al. (2006) were successful in cloning the mpd gene from a

Chlorpyrifos-degrading bacterium and using it for bioremediation of contaminated

soil. The gene encoding the organophosphorus hydrolase was cloned using a PCR

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cloning strategy based on the known Methyl parathion degrading (mpd) gene of

Plesiomonas sp. M6. Sequence BLAST result indicated that this gene has 99% similar

to mpd.

Zhang et al. (2005) placed the methyl parathion hydrolase (MPH)-encoding

gene mpd under the control of the P43 promoter and Bacillus subtilis nprB signal

peptide-encoding sequence. High- level expression and secretion of mature, authentic

and stable MPH were achieved using the protease-deficient strain B. subtilis WB800

as the host.

Plesiomonas spp. strain M6, which was capable of hydrolyzing methyl

parathion at high efficiency was isolated (Cui et al., 2001), and the gene (mpd)

encoding a novel methyl parathion hydrolase (MPH) was cloned (Cui et al., 2001)

and expressed in Escherichia coli (Fu et al., 2004). However, the previous work

showed that E. coli could process only a small proportion of the inactive precursor

polypeptide, comprising the signal peptide and the mature enzyme, to produce the

active MPH (Fu et al., 2004).

Guha et al. (1997) reported the plasmid borne genes of a soil bacterium

Micrococcus to be responsible for Chlorpyrifos degradation. The opd gene (including

its native signal peptide) was PCR amplified from pOP131 by Richins et al. (1997).

2.10. Genes involved in the degradation of Endosulfan

To identify the gene responsible for degradation of Endosulfan; Endosulfan

degrading Esd expression construct in pET14b (pET14b-esd-plasmid vector) was

used as positive control for detection of the specific gene in the strain ED1 isolated by

Vijaiyan Siva and Rajam (2013). Dot blot showed the positive expression of

Endosulfan degrading genes in the strain ED1.

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The metabolizing potential of a bacterial strain Rhodococcus MTCC 6716,

isolated from the gut of an Indian earthworm (Metaphire posthuma) was studied for

Endosulfan bioremediation by Verma et al. (2011). Endosulfan induced alterations in

the expression of mRNA and protein of specific Endosulfan metabolizing marker

gene (Esd) was studied. Endosulfan degradation was mediated through gene(s)

present in genomic DNA. Expression of marker gene was found to be Endosulfan

concentration dependent.

Weir et al. (2006) described the isolation of an Arthrobacter species capable

of degrading both isomers of the organochlorine insecticide Endosulfan and its toxic

metabolite, endosulfate. A gene, ese, encoding an enzyme capable of degrading both

isomers of Endosulfan and endosulfate was isolated from this bacterium. The ese gene

was located in a cluster of 10 open reading frames encoding proteins with low levels

of sulfur-containing amino acids. These open reading frames were organized into two

apparent divergently orientated operons and a gene encoding a putative LysR-type

transcriptional regulator. The operon not containing ese did contain a homologue

whose product exhibited 62% amino acid identity to the ese-encoded protein.

Sutherland et al. (2002b) studied the gram-positive bacterium Mycobacterium

sp. strain ESD, which was able to use Endosulfan as a source of sulfur for growth. A

cosmid library of strain ESD DNA was constructed in a Mycobacterium-Escherichia

coli shuttle vector and screened for Endosulfan-degrading activity in Mycobacterium

smegmatis, a species that did not degrade Endosulfan. Using this method, a single

cosmid was identified that conferred sulfur-dependent Endosulfan-degrading activity

on the host strain. An open reading frame (esd) was identified within this cosmid that,

when expressed behind a constitutive promoter in a mycobacterial expression vector,

conferred sulfite and sulfate- independent β-Endosulfan degradation activity on the

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recombinant strain. The translation product of this gene (Esd) had up to 50% sequence

identity with an unusual family of monooxygenase enzymes that use reduced flavins,

provided by a separate flavin reductase enzyme, as cosubstrates. An additional partial

open reading frame was located upstream of the Esd gene that had sequence

homology to the same monooxygenase family. A flavin reductase gene, identified in

the M. smegmatis genome, was cloned, expressed and used to provide reduced flavin

mononucleotide for Esd in enzyme assays.

Though there are literatures available on bacteria capable of degrading

Chlorpyrifos and Endosulfan, with the aim of obtaining efficient strains capable of

degrading the two pesticides and to use the cultures for degradation of combination of

pesticides the present topic has been taken up with the following objectives.

Isolation, screening and identification of bacteria capable of degrading

organophosphate (Chlorpyrifos) and organochlorine (Endosulfan) pesticides from

soil contaminated with the same by enrichment culture technique

Optimization of degradation of pesticides by respective cultures

Studies on the efficiencies of immobilized bacteria in pesticide degradation

Degradation of individual and combination of pesticides by individual and

consortium of bacteria

Extraction and analysis of pesticides and their degradation products by TLC and

LC-MS studies

Toxicity assessment of degradation products

Plasmid profile, plasmid curing experiments, enzymatic degradation studies and

PCR analysis

Pot studies to carry out bioformulation and bioremediation of soil