Propachlor Removal by Pseudomonas Strain GCH1 in an Immobilized-Cell System

Abstract

A bacterial strain capable of growing on propachlor (2-chloro-N-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genus Pseudomonas. Washed-cell suspensions of strain GCH1 accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 ؎ 0.15 mol h ؊1 . Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 ؎ 0.02 mg of protein mol ؊1 . GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.Propachlor (2-chloro-N-isopropylacetanilide) is an acylanilide herbicide widely used with corn, onion, cabbage, rose bushes, and ornamental plants. Microbial degradation (11,15,16,19) is the primary mechanism of acylanilide dissipation from soil. We previously reported the isolation of Pseudomonas strain PEM1 (3, 10, 11), which metabolizes the herbicide propachlor, yielding N-isopropylacetanilide, acetanilide, and acetamide as intermediates, and the isolation of Acinetobacter strain BEM2, which follows a different pathway and yields N-isopropylaniline and isopropylamine as intermediates. Lee et al. (9) reported that N-isopropylaniline, N-isopropylacetanilide, N-(1-hydroxyisopropyl)acetanilide, and N-isopropyl-2-acetoxyacetanilide were formed in soil treated with propachlor. Villareal et al. (18) proposed a propachlor degradative pathway yielding 2-chloro-N-isopropylacetamide and catechol as intermediates.Groundwater and soil contamination by herbicides has recently become of increasing concern (6,7,20). The relatively new concept of bioremediation provides a potentially cheap alternative to traditional disposal techniques, in addition to representing a genuine removal of contaminants by microbial degradation rather than the relocation of contaminants in such processes as landfilling. The problems presented by continuous fermentation processes could be resolved by the use of immobilized cells as biocatalysts (5,7,12,21).The aim of this study was to characterize the propachlor metabolism of a strain (GCH1) isolated from soil and to test a bioremediation system using immobilized cells in a reactor operating over a period of 150 days. The kinetics of the degradation and the viability of the immobilized cells are reported. MATERIALS AND METHODSIsolation of bacteria. Ten soil samples (10) were collected from agricultural fields in Madrid, Spain, with a history of propachlor contamination. Minimal medium (MB) (4) supplemented with 45 mg of propachlor liter Ϫ1 was inoculated with 20 g of soil sample and incubated at 28°C without sh...

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