Physiological and molecular characterization of organophosphate pesticide (profenofos and chlorpyrifos) degrading bacterial strains

Abstract

Organophosphate pesticides (OPs) are the synthetic chemicals that have broad applications in agriculture for controlling different kinds of pests such as insects and weeds etc. They poison the insects and mammals by paralyzing their central nervous system which is linked to many acute and long term health disorders. Two of the most widely used and broad-spectrum OP pesticides are the chlorpyrifos (CP) and profenofos (PFF) which are used for protecting various crops against serious insect pests. However, continuous and indiscriminate use of these pesticides is of great concern due to their serious impacts and hazards on the environment and humans. Remediation of these toxic pesticides and related contaminants using microorganisms having the right metabolic pathways seems to be the most effective technology. Objectives of this study were to isolate and characterize bacterial strains capable of complete degradation of CP, PFF and their toxic metabolites, optimize culture conditions that govern degradation of these compounds by the isolated bacteria and investigate the pathways of degradation. A chlorpyrifos degrading bacterial strain, Mesorhizobium sp. HN3 was isolated and characterized. Time course shake flask experiments and kinetic analysis revealed high efficiency of Mesorhizobium sp. HN3 for CP degradation up to 300 mg/L at range of at a broad range of culture conditions. Importantly, HN3 also degraded 3,5,6 trichloro-2- pyridinol (TCP), a more toxic and persistent metabolite of CP. Further, enhanced CP degradation in soil was achieved by the combined use of Mesorhizobium sp. HN3 and ryegrass (Lolium multiflorum). Moreover, a yfp-tagged variant of Mesorhizobium sp. HN3 (HN3yfp) was used to study the colonization of this strain in the rhizosphere and endosphere of ryegrass. The strain HN3yfp proficiently colonized the rhizosphere & roots of ryegrass, removed CP and TCP residues uptaken by the plant thus enhanced plant growth. For PFF degradation, a bacterial consortium PBAC, consisting of Achromobacter xylosoxidans, Pseudomonas aeruginosa, Bacillus sp. and Citrobacter koseri, was isolated. PBAC was capable of degrading PFF and its toxic hydrolysis product 4-bromo- 2-chlorophenol (BCP). The efficacy of PFF degradation was modeled by central composite design (CCD) based on response surface methodology (RSM). The simultaneous effects of three test interacting factors on the PFF degradation (%) were xvi monitored and conditions were optimized for maximum degradation of PFF. Gas Chromatography Mass Spectrometry (GC-MS) analysis of CP and PFF provided plenty of information regarding their metabolites and hence biodegradation pathways of the two pesticides were predicted successfully. The detection of dehalogenation and ring cleavage metabolites of the pesticides indicated the complete degradation of the toxic pesticides. The overall study indicates that CP degrading Mesorhizobium sp. HN3 and PFF degrading bacterial consortium PBAC are the promising candidates for the remediation of OP contaminated sites. Further, the study provides insight into the fate and biodegradation pathways of the two pesticides. Validity of the study is that fate of TCP or BCP have seldom been addressed. Rather, previous reports emphasis on the parent compound degradation. But the degradation of the metabolites is more important due to the fact that OP pesticides degrade to their metabolites soon after they reach soil. Metabolites are usually more toxic and persistent than the parent compounds. Moreover, to best of our knowledge this is the first study involving the elaborately designed optimization experiments for profenofos degradation by a diverse bacterial consortium. Also, degradation of BCP by the microbial communities has not already been reported.