Optimization of Growth Conditions for Chlorpyrifos-Degrading Bacteria in Farm Soils in Nakuru County, Kenya

Abstract

Chlorpyrifos (CP) is a chlorinated organophosphate pesticide. In Kenya, it is commonly used as an acaricide, particularly in dairy farming, leading to soil and water contamination. The study is aimed at isolating bacteria with CP-degrading potential and optimizing their growth conditions, including temperature, pH, and CP concentration. The enrichment culture technique was used, with minimal salt medium (MSM) supplemented with commercial grade CP. A multilevel factorial design was used to investigate the interactions of temperature, pH, and CP concentration. According to the findings, seven bacterial strains with potential to degrade CP were characterized and identified as Alcaligenes faecalis, Bacillus weihenstephanensis, Bacillus toyonensis, Alcaligenes sp. strain SCAU23, Pseudomonas sp. strain PB845W, Brevundimonas diminuta, and uncultured bacterium clone 99. Growth and biodegradation of bacteria differed significantly among the isolates across pH value, temperature, and concentrations (P ≤ 0.05). The optimum conditions for growth were pH 7, temperature of 25°C, and 25mg/l chlorpyrifos concentration, while optimum degradation conditions were pH 5, temp 25°C, and CP conc. 25mg/l. The Pearson correlation between optimum growth and degradation showed a weak positive relationship (R = 0.1144) for pH and strong positive relationship for temperature and concentration of chlorpyrifos. Other than pH, the study shows that there could be other cofactors facilitating the chlorpyrifos degradation process. The findings show that an efficient consortium, at 25°C and pH 5, can include Bacillus toyonensis 20SBZ2B and Alcaligenes sp. SCAU23 as they showed high optical density (OD) values under these conditions. These results indicate the potential for these bacteria to be employed in chlorpyrifos-contaminated ecosystem detoxification efforts upon manipulation of natural growth conditions. The findings of this study offer a potential foundation for future research into the reconstitution of a consortium. Based on the optimum conditions identified, the isolated bacterial strains could be further developed into a consortium to effectively degrade CP in both laboratory and field conditions. Dairy farmers can utilize the isolated strains and the consortia to decontaminate farm soils.

Figure 1

Tetrazolium test results showing color change to purple in all 7 isolates. The image on the top left shows the setup before the experiment. Images MW1 to MW7 show the change in color in the inoculated tubes to purple (due to degradation of CP), while the control remained colorless, indicating no degradation. Key: MW1: Alcaligenes faecalis UWI9; MW2: Bacillus weihenstephanensis FB25M; MW3: Bacillus toyonensis 20SBZ2B; MW4: Alcaligenes sp. SCAU23; MW5- Pseudomonas sp. P_B845W; MW6: uncultured bacterium clone 99; MW7: Brevundimonas diminuta CB21.

Figure 2

Growth patterns of bacteria isolates (OD₆₀₀) and CP degradation (OD₄₈₀) at pH 5 (a), pH 7 (b), and pH 9 (c). MW1: Alcaligenes faecalis UWI9; MW2: Bacillus weihenstephanensis FB25M; MW3: Bacillus toyonensis 20SBZ2B; MW4: Alcaligenes sp. SCAU23; MW5: Pseudomonas sp. P_B845W; MW6: uncultured bacterium clone 99; MW7: Brevundimonas diminuta CB21. Optical density at 600 nm was used for indirect measurement of bacterial growth, while optical density at 480 nm was to measure the CP degradation as indicated by the intensity of formazan.

Figure 3

Growth patterns of bacteria isolates (OD₆₀₀) and CP degradation (OD₄₈₀) at 25°C (a), 30°C (b), and 37°C (c). MW1: Alcaligenes faecalis UWI9; MW2: Bacillus weihenstephanensis FB25M; MW3: Bacillus toyonensis 20SBZ2B; MW4: Alcaligenes sp. SCAU23; MW5: Pseudomonas sp. P_B845W; MW6: uncultured bacterium clone 99; MW7: Brevundimonas diminuta CB21. Optical density at 600 nm was used for indirect measurement of bacterial growth, while optical density at 480 nm was used to measure the CP degradation as indicated by the intensity of formazan, a product of tetrazolium breakdown by the bacterial isolates.

Figure 4

Growth patterns of bacteria isolates (OD₆₀₀) and CP degradation (OD₄₈₀) at 25 mg/l (a), 50 mg/l (b), and 100 mg/l (c). The bacteria isolates are as follows: MW1: Alcaligenes faecalis UWI9; MW2: Bacillus weihenstephanensis FB25M; MW3: Bacillus toyonensis 20SBZ2B; MW4: Alcaligenes sp. SCAU23; MW5: Pseudomonas sp. P_B845W; MW6: uncultured bacterium clone 99; MW7: Brevundimonas diminuta CB21. Optical density at 600 nm was used for indirect measurement of bacterial growth, while optical density at 480 nm was used to measure the CP degradation as indicated by the intensity of formazan, a product of tetrazolium breakdown by the bacterial isolates.

Figure 5

(a) Correlation between bacterial growth and CP degradation at optimum pH 5. (b) Correlation between bacterial growth and CP degradation at optimum temperature 25°C. (c) Correlation between bacterial growth and CP degradation at optimum concentration 25 mg/l.