Biodegradation of chlorpyrifos and its hydrolysis product 3,5,6-trichloro-2-pyridinol using a novel bacterium Ochrobactrum sp. JAS2: A proposal of its metabolic pathway

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Highlights

  • The strain JAS2 was identified as Ochrobactrum sp. which was capable of degrading chlorpyrifos and its metabolite TCP.

  • The biodegradation pathway of chlorpyrifos has been proposed.

  • Further JAS2 strain was found to possess auxiliary plant growth promoting activities.

Abstract

Biodegradation of chlorpyrifos and its major metabolite 3,5,6-trichloro-2-pyridinol (TCP) were studied with a novel bacterial strain JAS2 isolated from paddy rhizosphere soil. The molecular characterization based on 16S rRNA gene sequence homology confirmed its identity as Ochrobactrum sp. JAS2. The JAS2 strain degraded 300 mg l 1 of chlorpyrifos within 12 h of incubation in the aqueous medium and it produced the TCP metabolite. However, after 72 h of incubation TCP was also completely degraded by the JAS2 strain. A tentative degradation pathway of chlorpyrifos by Ochrobactrum sp. JAS2 has been proposed on basis of GC–MS analysis. The complete degradation of chlorpyrifos occurred within 24 h in the soil spiked with and without addition of nutrients inoculated with Ochrobactrum sp. JAS2. TCP was obtained in both the studies which was degraded completely by 96 h in the soil spiked with nutrients and whereas 120 h in absence of nutrients in the soil. The mpd gene which is responsible for organophosphorus hydrolase production was identified. The isolates Ochrobactrum sp. JAS2 also exhibited a time dependent increase in the amount of tricalcium phosphate solubilization in Pikovskaya's medium. Further screening of the strain JAS2 for auxiliary plant growth promoting activities revealed its remarkable capability of producing the indole acetic acid (IAA), hydrogen cyanide (HCN) and ammonia.

Introduction

The moderately hazardous organophosphate insecticide chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate] is commonly used in India [1]. Chlorpyrifos is a non-systemic insecticide, acaricide, and termiticide against gall midge, cutworms, corn rootworms, leaf folder, leaf hopper, etc. which is effective against a wide range of insect pests of economically important crops [2]. It enters into the insect body by contact and ingestion, and is also absorbed through the gut, skin and pulmonary membranes [3]. Usually, it affects the nervous system of the target insects by inhibiting the activity of acetylcholinesterase by phosphorylation, both at the synapse of neurons and in the plasma [4]. As a result, acetylcholine is accumulated at the neuron synapse which causes the death of the target insect. The half-life of chlorpyrifos in soil is usually between 10 and 120 days, but can range from 2 weeks to over one year, depending on the soil type, climate, and other conditions [5].

The biodegradation of chlorpyrifos results in the formation of TCP, which is the primary and major degradation product. It has greater water solubility than chlorpyrifos and causes the widespread contamination in soils and in the aquatic environment. TCP is not only persistent towards degradation by microorganisms but also affects the biodegradation of chlorpyrifos owing to its antimicrobial activities [6]. Living organisms has been exposed to pesticide residues in soil and water, resulting in a risk to the ecological balance [7]. The remediation of chlorpyrifos contaminated sites to mitigate the hazardous effects of such toxic chemicals is required. Biotic degradation is one of the most viable options for the remediation of chlorpyrifos in soil and water. Several researchers have focused on the microbial degradation which has been reported as a primary mechanism of pesticide dissipation from the soil and water environment. To date, few microorganisms capable of degrading chlorpyrifos and its metabolite TCP have been isolated. Such microorganisms include, Alcaligenes faecalis strain DSP3 degraded 100% and 93.5% of 100 mg l 1 chlorpyrifos and TCP within 12 days [8], and Sphingomonas sp. strain Dsp-2 degraded 100 mg l 1 chlorpyrifos within 24 h, but only 30 mg l 1 of TCP within 48 h [9]. Paracoccus sp. TRP degraded 50 mg l 1 chlorpyrifos and TCP in 4 days [10] and Bacillus pumilus C2A1 degraded 89% of 1000 mg l 1 chlorpyrifos within 15 days, and 90% of 300 mg l 1 TCP within 8 days [11]. Cupriavidus sp. DT-1 degraded completely 100 mg l 1 chlorpyrifos within 6 h and 100% of 50 mg l 1 TCP within 14 h [12]. To the best of our understanding, very few reports are available on the microorganisms with concurrent plant growth promoting abilities besides their role in pesticide degradation [13], [14], [15]. Thus, the prime objective of the present investigation was to screen the chlorpyrifos and its metabolite TCP resistant bacteria isolate from paddy rhizospheric soil for selection of an efficient strain with inherent chlorpyrifos degrading ability, and multiple auxiliary plant beneficial traits.

Section snippets

Chemicals

Analytical grade chlorpyrifos methyl PESTANAL (99.9%) and TCP (99.3%) were procured from Sigma-Aldrich (St. Louis, MO, USA). The pesticide used in this study of technical grade chlorpyrifos 20% (EC) purchased from Isagro (Asia) Agrochemical Pvt. Ltd., Mumbai. All other reagents used in this study were of high purity and analytical grade.

Isolation and characterization of chlorpyrifos and its metabolite TCP degrading bacterial strain

Bacterial strains capable of degrading chlorpyrifos were isolated from paddy rhizosphere of chlorpyrifos treated soil by the enrichment culture technique. 20 g

Isolation and characterization of strain JAS2

The chlorpyrifos degrading bacterium JAS2 is a Gram-negative, rod shaped, motile bacterium, positive for citrate, catalase, oxidase, urease, nitrate reduction, glucose, sucrose, arabinose, mannitol and rhamnose, negative for indole, methyl red, Voges–Proskauer, H2S production, starch hydrolysis, gelatine liquefaction, lactose and sorbitol. Comparative analysis of the 16S rRNA gene sequence of strain JAS2 illustrated 98% similarity with the species Ochrobactrum oryzae of the genus Ochrobactrum

Conclusion

A novel chlorpyrifos degrading bacterium was isolated and characterized as Ochrobactrum sp. JAS2 with the unique capability of degrading chlorpyrifos and its major metabolite TCP with concurrent plant growth promoting and biocontrol potential. This is the first report involving biodegradation of both chlorpyrifos and TCP by a bacterial strain from the genus Ochrobactrum. The present study offers successful technology for the development of a powder based bioformulation. The ability to degrade

Acknowledgment

This research work was funded by the Department of Science and Technology (DST), Govt of India, New Delhi. Research grant sanction no. DST/TSG/NTS/2009/67.

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