Microbial emulsification and metabolism of organophosphorous pesticides.

Abstract

The ever-increasing usage of pesticides during the last three decades has caused widespread environmental contamination. For this reason, the more persistent organochlorine pesticides have been replaced by less persistent organophosphorus, carbamate, and other new-generation pesticides. Organophosphorus pesticides, which form a large and diverse group, are susceptible to chemical and biological reactions, especially microbial, in the environment. However, complete microbial degradative pathways for these pesticides have not been established, mainly because of their low miscibility/solubility in water and their toxic nature.

The problem of low miscibility and solubility in aqueous medium has been partly overcome either by formation of soluble sodium salts or by the use of detergents. Many microorganisms produce surface-active agents (bioemulsifiers and biosurfactants) when they utilize insoluble or immiscible compounds such as hydrocarbons. Emulsification of 2,4,5-trichlorophenoxyacetic acid and related chlorinated compounds has been reported. No microbial bioemulsifier had previously been described for insoluble or immiscible organophosphorus pesticides. The present investigation describes an emulsifying activity specific for immiscible organophosphorus pesticides (e.g., fenthion, fenitrothion).

During attempts to isolate bacteria capable of utilizing immiscible organophosphorus pesticides as sole carbon and energy sources, two bacterial strains capable of emulsifying these pesticides were isolated from a fenthion enrichment culture. The strains were identified as divergent forms of Bacillus subtilis based on morphological, nutritional, and biochemical characteristics. Emulsifying activity from one strain, Bacillus subtilis FE-2, was studied. The activity is expressed constitutively and released from the cell surface during optimal growth only when the pesticide is present in the medium under high agitation.

The emulsifying activity is nondialyzable, heat-resistant, and functions most efficiently between pH 7.0 and 7.6. It is specific for immiscible organophosphorus pesticides. The emulsifying agent is sensitive to lysozyme and SP-249, partially sensitive to some proteases and phospholipase C, and can be extracted by solvents and precipitated by trichloroacetic acid or ammonium sulphate. It appears to be a macromolecule consisting of carbohydrate, protein, and lipid.

The macromolecular bioemulsifier does not require metal ions or small molecular weight dialyzable factors. Treatment with lysozyme results in loss of emulsifying activity, and a protein band of molecular weight 43,000 disappears, indicating this protein possesses biological (emulsifying) activity. Based on its interaction with lysozyme, the emulsifier appears to be ionic in nature.

The emulsifier was purified to near homogeneity by ion-exchange and gel-filtration chromatography. It consists of a single subunit type with an apparent molecular weight of 43,000. Low yields, typical of microbial emulsifiers, prevented further purification and detailed chemical/structural analysis.

Bacillus subtilis FE-2 metabolizes fenitrothion by more than one pathway—reductive and hydrolytic. Apart from 3-methyl-4-nitrophenol (hydrolysis product) and amino-fenitrothion (reduction product), other chloroform-soluble and highly polar metabolites are formed. However, rates of fenitrothion metabolism are low, especially via the hydrolytic pathway. Only low levels of fenitrothion hydrolase activity are detected, and negligible amounts of ^14CO2 are generated from [U-^14C] fenitrothion. Thus, in Bacillus subtilis FE-2, there is no correlation between its ability to emulsify and its extent of degradation of immiscible organophosphorus pesticides. The exact role of the emulsifier for the producing strain remains unknown.

Industrial Potential: The emulsifier has potential applications in pesticide formulations and disposal. It could be exploited for removal and recovery of pesticides and in controlled, more efficient delivery systems.