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. 2022 Mar 24;8(3):e09176.
doi: 10.1016/j.heliyon.2022.e09176. eCollection 2022 Mar.

Molecular characterization of chlorpyrifos degrading bacteria isolated from contaminated dairy farm soils in Nakuru County, Kenya

Micah Nyabiba Asamba  1 , Ezekiel Njeru Mugendi  1 , Paul Sifuna Oshule  2 , Suliman Essuman  3 , Lenny Mwagandi Chimbevo  4 , Norbert Adum Atego  4
Affiliations

Molecular characterization of chlorpyrifos degrading bacteria isolated from contaminated dairy farm soils in Nakuru County, Kenya

Micah Nyabiba Asamba et al. Heliyon. .

Abstract

Chlorpyrifos (CP) is an organophosphate widely used as an insecticide and acaricide. Extensive application of CP contaminates ecosystems, polluting the environment and food products, creating health complications to humans due to its neurotoxicity. The study evaluated CP bioremediation by bacteria isolated from dairy farm soils in Nakuru County, Kenya, through enrichment culture technique. The growth response of the bacteria and degradation of chlorpyrifos was monitored every five days using UV-VIS Spectrophotometer (600nm). Enrichment culture technique led to the isolation of eighteen (MA1-MA18) potential CP degraders belonging to the genera Pseudomonas, Stenotrophomonas, Bacillus, Alicaligenes, and Achromobacter. The efficacy of four (4) strains was further investigated using Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The results showed that all four strains significantly degraded chlorpyrifos in Minimum Salt Medium (MSM): Lysinibacillus sp.HBUM206408 (87.16 %), Stenotrophomonas maltophilia (82.04 %), Pseudomonas putida (89.52 %), and Achromobacter insuavis (91.08 %) within 16 days, producing 2-Hydroxy-3, 5, 6-trichloropyridine (TCP) as the main metabolite. Therefore, these strains can be used to degrade chlorpyrifos in contaminated soil. There is a need for further studies to determine the possible mechanisms and other metabolites of chlorpyrifos degradation by the isolates obtained in the study. Besides, future studies should explore the efficacy and survival of the organisms in the contaminated environment.

Keywords: Biodegradation; Bioremediation; Chlorpyrifos; Metabolite; Organophosphate; Spectrophotometer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Map of the study area, Nakuru County, Kenya.
Figure 2
Figure 2
Microbial growth curves of chlorpyrifos-enriched MSM cultures (A- 5 ppm; B- 10 ppm; and C- 40 ppm). Abbreviations: N1S- Nakuru soil 1; N2S- Nakuru soil 2; N3S- Nakuru soil 3; N1D- Nakuru Dip wash 1; N2D-Nakuru Dip wash 2; and N3D- Nakuru Dip wash 3. Control- Uninoculated CP-supplemented MSM media.
Figure 3
Figure 3
Nutrient Agar Plates of Isolated CP degrading bacteria (identified as A- Achromobacter insuavis, B- Lysinibacillus sp., C- Pseudomonas protegens, D- Alcaligenes faecalis, E- Stenotrophomonas maltophilia, and F- Pseudomonas putida).
Figure 4
Figure 4
PCR amplified 16S rDNA of the bacteria isolates in agarose gel. Lane M, 1kb DNA ladder; Lanes 1–18, genomic DNA of isolated strains (MA1 - MA18); Lane 19, Negative control. (The full, original gel images are provided in supplemental material as Agarose gel image of PCR products 1 and 2).
Figure 5
Figure 5
Phylogenetic tree showing the relationship of the 18 isolates (MA1 to MA18) to closely related bacteria. Reference type strains of corresponding bacteria are involved in the tree. Four isolates that were subjected to GC-MS analysis (MA1, MA2, MA4, and MA8 are underlined in blue.
Figure 6
Figure 6
GC chromatograms showing the retention time of CP at 18.87 min (A) and TCP 13.40 min (B) and other smaller unidentified metabolites.
Figure 7
Figure 7
Chemical structure and mass spectra of metabolite 3, 5, 6-trichloro-2-pyridinol (TCP) produced from chlorpyrifos degradation.
Figure 8
Figure 8
Degradation kinetics of CP by Isolates MA1, MA2, MA4, and MA8 as compared to the control.
Agarose Gel Image of PCR Products 1
Agarose Gel Image of PCR Products 1
Agarose Gel Image of PCR Products 2
Agarose Gel Image of PCR Products 2
:
See this image and copyright information in PMC

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