Unveiling six novel bacterial strains for fipronil and thiobencarb biodegradation: efficacy, metabolic pathways, and bioaugmentation potential in paddy soil

Abstract

Introduction: Soil bacteria offer a promising approach to bioremediate pesticide contamination in agricultural ecosystems. This study investigated the potential of bacteria isolated from rice paddy soil for bioremediating fipronil and thiobencarb, common agricultural pesticides.

Methods: Bacterial isolates capable of degrading fipronil and thiobencarb were enriched in a mineral salt medium. A response surface methodology with a Box-Behnken design was utilized to optimize pesticide degradation with the isolated bacteria. Bioaugmentation tests were performed in paddy soils with varying conditions.

Results and discussion: Six strains, including single isolates and their mixture, efficiently degraded these pesticides at high concentrations (up to 800 µg/mL). Enterobacter sp., Brucella sp. (alone and combined), and a mixture of Stenotrophomonas sp., Bordetella sp., and Citrobacter sp. effectively degraded fipronil and thiobencarb, respectively. Notably, a single Pseudomonas sp. strain degraded a mixture of both pesticides. Optimal degradation conditions were identified as a slightly acidic pH (6-7), moderate pesticide concentrations (20-50 µg/mL), and a specific inoculum size. Bioaugmentation assays in real-world paddy soils (sterile/non-sterile, varying moisture) demonstrated that these bacteria significantly increased degradation rates (up to 14.15-fold for fipronil and 5.13-fold for thiobencarb). The study identifies these novel bacterial strains as promising tools for bioremediation and bioaugmentation strategies to tackle fipronil and thiobencarb contamination in paddy ecosystems.

Keywords: bacteria; bioremediation; degradation rate; pesticide; response surface methodology; transformation products.

Figure 1

(A,B) Fipronil degradation and bacterial growth, respectively in MSM inoculated with FA, FB, and FM, (C,D) thiobencarb degradation and bacterial growth, respectively in MSM with TA, TB, TC, and TM, (E) fipronil + thiobencarb mixture degradation in MSM inoculated with MA, MB, MC, and MM. In the case of the fipronil + thiobencarb mixture, the initial turbidity of the culture, resulting from adding two pesticide solutions, prevented the measurement of OD₆₀₀. Error bars represent standard deviations (n = 3).

Figure 2

Relationship between initial concentrations of fipronil and thiobencarb and their specific degradation rates by the selected isolates and consortia. (A–C): degradation of fipronil concentrations by FA, FB, and FM, respectively, (D): degradation of thiobencarb concentrations by TM, (E,F): degradation of fipronil and thiobencarb, respectively by MA.

Figure 3

Response surface 3D graphs for fipronil degradation optimization by FA, FB, and FM. (A,D,G) effect of pesticide concentration and inoculum size (OD₆₀₀) on fipronil degradation by FA, FB, and FM, respectively, (B,E,H) effect of pH and inoculum size (OD₆₀₀) on fipronil degradation by FA, FB, and FM, respectively, (C,F,I) effect of pH and pesticide concentration on fipronil degradation by FA, FB, and FM, respectively.

Figure 4

Response surface 3D graphs for thiobencarb degradation optimization by TM. (A) Effect of pesticide concentration and inoculum size (OD₆₀₀) on thiobencarb degradation by TM, (B) effect of pH and inoculum size (OD₆₀₀) on thiobencarb degradation by TM, (C) effect of pH and pesticide concentration on thiobencarb degradation by TM.

Figure 5

Response surface 3D graphs for fipronil and thiobencarb degradation optimization by MA. (A,D) Effect of pesticide concentration and inoculum size (OD₆₀₀) on fipronil and thiobencarb degradation, respectively by MA, (B,E) effect of pH and inoculum size (OD₆₀₀) on fipronil and thiobencarb degradation, respectively by MA, (C,F) effect of pH and pesticide concentration on fipronil and thiobencarb degradation, respectively by MA.

Figure 6

Proposed degradation pathways of fipronil by FA and MA. Fipronil is either transformed to N-(Trifluoroacetyl)aminoacetic acid and 1-Pentanamine, N-pentyl- (pathway 1) or to 4-(Trifluoromethyl)-phenol and 1,4-Benzenediol, 2-methyl- (pathway 2). For MA, transformation to 1-Pentanamine, N-pentyl- was not observed.

Figure 7

Proposed degradation pathway of fipronil by FB. Fipronil is either transformed to 3H-1,2,4-Triazole-3-thione,2,4-dihydro-2,4,5-trimethyl- and Thiophene, 2-nitro- (pathway 1) or to Benzenamine, 2,4-dimethyl- and 1,4-Benzenediol, 2-methyl- (pathway 2).

Figure 8

Proposed degradation pathway of fipronil by FM. Fipronil was transformed directly to 1-Aminononadecane, N-trifluoroacetyl-, Heptadecanenitrile, and 2-hexadecanole (pathway 2) or via an intermediate metabolite, N-(Trifluoroacetyl)aminoacetic acid (pathway 1). Alternatively, FM degraded fipronil to 4-(Trifluoromethyl)-phenol and 1,4-Benzenediol, 2-methyl- (pathway 3).

Figure 9

Proposed degradation pathway of thiobencarb by TM and MA. Thiobancarb is either transformed to Benzenecarbothioic acid, S-methyl ester and Carbamothioic acid, diethyl-, S-ethyl ester (pathway 1) or to Benzothiazole, 2-methyl- (pathway 2). Ultimately, both pathways converge to form 1-Hexadecanethiol.