Isolation of Bacteria from Agricultural Soils and Evaluation of Their Degradative Capacity for Organochlorine and Organophosphorus Pesticides

Abstract

In this work, OP- and OC-degrading bacteria were isolated from agricultural soil samples taken in the department of Bolivar, Colombia. The objective of this research was to degrade organochlorine and organophosphorus pesticides using bacterial colonies native to agricultural soils. Two bacterial colonies were isolated from the soil samples, which showed a higher degree of adaptation to media contaminated with the pesticide mixtures. They were identified by biochemical tests using BBL Crystal kits, and, subsequently, their 16S rDNA was sequenced using the PCR technique. Bacterial growth was studied by the OD index, taking absorbance readings on a UV-VIS spectrophotometer at 600 nm, at the 0.5 McFarland scale, and quantification of pesticide degradation was studied by GC–MS. The colonies identified were Bacillus cereus and Paenibacillus lautus. B. cereus isolates were exposed to the OPs malathion, chlorpyrifos, and coumaphos [80 mg·L⁻¹], degrading at rates of 52.4%, 78.8% and 79.5%, respectively, after 12 days of incubation in liquid medium at pH = 7.0 ± 0.2 and 37 °C. Furthermore, P. lautus isolates exposed to the OCs lindane, metolachlor, endrin, and p,p′-DDT [80 mg·L⁻¹] degraded at rates of 64.0%, 60.8%, 55.7% and 65.1% under the same conditions of temperature, pH, and incubation time. These results show that B. cereus and P. lautus might be useful for cleaning up environments that have been polluted by OPs and OCs.

1. Introduction

The production of synthetic organic pesticides worldwide has increased from the early 20th century to the present day, due to the development of agribusiness and the high rate of demand for food. Sources of pesticide contamination can be croplands, atmospheric precipitation, and hazardous waste disposal sites. Pesticides can be transported by air over long distances [1]. Agricultural use of pesticides is a subset of the broader spectrum of industrial chemicals used by modern society, in which OPs and OCs are prominent [2]. OPs have been widely used in the protection of crops and agricultural and forestry resources, as well as for the control of vectors and disease-transmitting organisms, in which area they have achieved a high level of impact by reducing production losses in crops of considerable importance [3]. On the other hand, although OCs were banned decades ago, as they are very stable molecules, residues are still found in the soils on which they were used. Among their physicochemical properties, it should be noted that OCs, having a halide group, have high physical and chemical stability, while OPs, due to their phosphate group, are susceptible to metabolism, which increases their degree of toxicity [4,5].

These groups, OCs, and OPs, have similar properties, being insoluble in water, non-volatile, and highly soluble in organic solvents, which is conducive to their persistence in the environment [6]. These characteristics have led to their restriction and even prohibition in some regions. In Colombia, chlorinated insecticides were banned by Resolution 447 of 1974 of the Ministry of Agriculture, which is why they have been replaced by other less harmful and persistent substances, whose distribution and production can be more regulated [7]. Although most of these pesticides are not used today, their residues are still found in many environmental matrices because of bioaccumulation and biomagnification, causing environmental contamination [8,9,10,11]. One of the most studied terms associated with the presence of pesticides in the environment is their persistence, which is defined as the ability of pesticides to maintain their physicochemical and functional characteristics in the environment in which they are transported or distributed for a limited period after their release [12,13,14,15,16,17]. Several factors, such as hydrophobic porosity, microbial activity, moisture, pH, and co-existence with metals, affect the mass transfer of pesticides in soils [18,19,20].

In the literature, there are examples of pesticide degradation by microorganisms. Biodegradation, like biotransformation, refers to a process in which an organism transforms one chemical compound into another, which involves a series of reactions that occur under certain conditions and at different times, and are usually biochemical factors of the enzymatic activity of the organism. During this process, the biodegradable substance or material is reduced to its basic components [21]. Several factors affect or limit a biodegradation process. Among those that have been identified are the substrate selectivity of enzymes and all the possible biochemical mechanisms possessed by microorganisms, the chemical structure and complexity of the compound, and environmental factors such as pH, temperature, and humidity. However, this process allows the behavior of microorganisms in the presence of contaminants in their environment to be studied, as well as the concentration of these substances before, during, and after all the steps involved [22,23].

Bacterial colonies living in pesticide-contaminated environments can develop the ability to degrade pesticides, although the degradation pathways are limited by low enzymatic activity. To improve the catalytic activity and enzyme specificity of microorganisms for specific substrates, biotechnological techniques such as biostimulation, bioaugmentation, and surfactant addition have been applied [21,24]. In some cases, metabolites produced from the biotransformation of a contaminant remain toxic or resistant to other degradation processes. One approach to improve these degradation processes is the use of microorganisms that follow the required degradation pathways for each compound [25,26].

This is where understanding the physiology and genetics of the microbial populations involved in bioremediation processes is useful for assessing and enhancing biodegradation [27]. Advances in molecular biology methods have facilitated the study of microbial populations [28]. Recent advances in molecular biology techniques, such as quantitative real-time PCR, have made it possible to track and identify bacteria and catabolic genes involved in pesticide degradation. These techniques provide a picture of the relevant catabolic microorganisms and functional genes present in a contaminated system [29,30]. PCR amplification of 16S rDNA fragments is often used as a tool to determine temporal or spatial differences in bacterial populations and to monitor changes in gene diversity [28].

Microbial degradation of pesticides involves redox-type chemical reactions to gain energy from electron transfer between electron acceptors and electron donors. Microbes can also capture carbon, nitrogen, or trace elements to build their cellular structures [31,32,33].

Considering all the factors that affect OC and OP pesticides at the environmental level, new methodologies have been developed for the remediation of the damage caused by the improper use of these pesticides, where research involves the use of living organisms, microorganisms, and/or enzymes that contribute to the detoxification of contaminants in different environmental matrices [33,34,35,36,37,38,39,40]. Depending on their fate, these organophosphate compounds can become bioavailable for microbial degradation. There are several studies that show the versatility of different species of bacteria in degrading pesticides, including organophosphorus and/or organochlorines [32,33,34,35,36,37,38]. Among the species of bacteria that have been reported as biodegraders of OP pesticides are Pseudomonas putida [39,40,41,42], Kocuria assamensis [43], Micrococcus sp. [44], Bacillus [27,45,46,47,48,49,50,51,52], Enterobacter cancerogenus, Pseudomonas aeruginosa [53], Agrobacterium tumefaciens, Cellulosimicrobium funkei, Shinella zoogloeoides, and Bacillus aryabhattai [54], Serratia sp. [55,56], Enterococcuslectis, Enterobacter cloacae, Stenotrophomonas maltophilia, and Escherichia coli [42].

On the other hand, there is also research on the biodegradation of chlorinated pesticides, for example, the remediation of endosulfan by the indigenous Pseudomonas sp. [57]. Bacteria isolated from coffee beans, P. aeruginosa, and F. oryzihabitans were selected for DDT and endosulfan degradation [58]. Ochrobactrum sp. and Microbacterium oxydans sp. were employed in the degradation of β-HCH [59].

The scientific literature shows that indigenous microorganisms are normally preferred over exogenous strains, to avoid altering natural microbial diversity and protect competitiveness in the local environment, and microbial degradation is an efficient method to inhibit chemical contamination of soil [60].

Therefore, the objective of this work was to degrade organochlorine and organophosphorus pesticides using native bacterial strains from agricultural soils, as a contribution to the global study of microbial strategies to improve soil fertility and promote sustainable land management practices.

2. Materials and Methods

2.1. Soil Sample Collection

Soil samples were collected in the municipality of San Joaquín, Mahates, located in the department of Bolívar (Colombia) (10°09′42″ N; 75°06′41″ W) (Figure 1). According to the procedure described by Brady and Weil (2017) [61], three (3) samples, cataloged as L1, L2, and L3, were taken at different sampling sites separated at distances between 500 and 600 m, to cover the largest possible area. Each sample consisted of thirteen (13) subsamples collected in a zigzag pattern at a depth of 20 cm, after removing the topsoil. Once the subsamples were collected, they were combined as a single sampling site (L). Finally, they were stored in sterilized, hermetically sealed bags at ≤20 °C to retain as much moisture as possible.

2.2. Chemicals and Microbial Culture Medium

Analytical pesticide standards of purity ≥95% (malathion, chlorpyrifos, lindane, metolachlor, endrin, and p,p′-DDT) were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Coumaphos (99.4 % purity) was obtained from Sigma-Aldrich (St Louis, MO, USA). Stock solutions of the pesticides (200 and 100 mg·L⁻¹) were prepared in hexane (Merck KgaA, Darmstadt, Germany).

The nutrient composition of the minimal culture medium M9 used for bacterial enrichment was (NH₄)₂SO₄ 2 g·L⁻¹, MgSO₄·7H₂O 0.25 g·L⁻¹, CaCl₂·2H₂O 0.01 g·L⁻¹, FeSO₄·7H₂O 0.01 g·L⁻¹, NaH₂PO₄·7H₂O 1.5 g·L⁻¹, and KH₂PO₄ 1.5 g·L⁻¹, and it was neutralized at pH 7.1 ± 2H₂SO₄ 2M and distilled water, after sterilization. LB Agar (Luria–Bertani, Merck KgaA, Darmstadt, Germany) was used for the isolation and growth of isolates.

2.3. Preparation of Soil Samples and Activation of Pesticide-Degrading Metabolism in Bacterial Colonies

Establishing a biostimulation protocol, soil samples L1, L2, and L3 were sieved with a 1.5 mm sieve, and from each of these, approximately 150 g was taken. In parallel, standard mixtures of each pesticide group were prepared; for OPs, a mixture of malathion, chlorpyrifos, and coumaphos was used, and in the case of OCs, a mixture of lindane, metolachlor, endrin, and p,p′-DDT was used, at a concentration of 200 mg·L⁻¹ in hexane. A 10 mL volume of each of these solutions was taken and added to the screened soils. Finally, soil samples treated with the pesticides were stored in closed glass containers, in a dry, dark place, at room temperature for 20 days [53].

2.4. Isolation of Native Bacterial Colonies from Agricultural Soils

A mass of 1 g of each soil sample treated with one of the mixtures of pesticide standards was diluted in 10 mL of M9 minimal culture medium; subsequently, a 1 mL aliquot was transferred to a test tube containing 9 mL of minimal medium, and at least 3 dilutions were serially repeated in 1:10 v/v ratios and incubated for 7 days at 37 °C. Petri dishes, 90 mm in diameter, containing LB agar were inoculated with these solutions using the streaking technique, and after 24 h, under the same incubation conditions, the most abundant bacterial colonies were isolated from the samples obtained. Finally, the selected colonies were purified and preserved in thioglycolate glycerol broth at ≤20 °C.

2.5. Biochemical and Molecular Identification of Isolated Bacterial Colonies

The representative (most abundant) colonies isolated and purified were subjected to conventional microbiological tests, such as Gram-staining, oxidase, and catalase. For species identification, two procedures were carried out: biochemical identification and molecular identification. Biochemical identification was performed with specialized BBL Crystal kits (Becton, Dickinson and Company, Sparks, MD, USA) [62]. It was compared with molecular identification, in which DNA sequencing was obtained by isolation and purification of the 16S ribosomal gene by the polymerase chain reaction (PCR) technique, and the purification of the PCR amplified fragments, and their sequencing was performed by the Sanger method, using primers 337F, 518F, 800R, and 1100R of the 16S gene. The taxonomic analysis of the sequences obtained was performed using the BLAST tool (Basic Local Alignment Search Tool) of the NCBI (National Center for Biotechnology Information) (http://www.ncbi.nlm.nih.gov/blast/ accessed on 19 July 2019), comparing against the reference RNA database “refseq_rna”, using the “Classifier” and “SeqMatch” tools on the RDP (Ribosomal Data Project) website.

2.6. Quantification of Pesticide Concentration and Determination of Bacterial Biomass, During Biodegradation Process

To separate vials, 9.8 mL of M9 medium and 100 µL of the pesticide mixture [200 mg·L⁻¹] were added, and shaken in a vortex until homogenized; subsequently, 100 µL of the bacterial suspension was added, at the 0.5 McFarland scale, striving for a biomass of approximately 1.5·10⁸ CFU·mL⁻¹ and a pesticide concentration of 80 mg·L⁻¹. These vials were incubated for different periods, for up to 12 days, at 37 °C, and at a pH between 7.0 and 7.2, with continuous shaking. Every 24 h, OD readings (λ = 600 nm) were taken on a Thermo Evolution^TM 201 UV-VIS spectrophotometer (Madison, WI, USA) using glass cells, to determine the biomass of bacterial growth [46]. Liquid–liquid extractions were then performed at 1:1 volume with dichloromethane; the obtained organic phase was preconcentrated in an rotary evaporator (IKA BH 10 Control, Werke, Germany), the solvent was dried with N₂, and the volume was restored with hexane, and the aliquot was filtered through an anhydrous sodium sulfate to remove possible water contents. Finally, the samples were analyzed on an Agilent 7890A gas chromatograph (Agilent Co., Palo Alto, CA, USA) coupled to an Agilent MSD 5975c mass spectrometry detector (GC–MS). They were analyzed using an HP-5 column (350 °C, 30 m × 250 µm × 0.25 µm). The method had limits of detection (LODs) of 0.01–0.04 μg/L and limits of quantification (LOQs) of 0.04–0.13 μg/L. To quantify the change in the pesticides over time as a result of the degradation process, the analysis conditions were an initial temperature of 60 °C, with a ramp of 15 °C/min, up to a temperature of 290 °C, using He as a carrier gas, with a flow rate of 1.0 mL/min; the MS was kept in full scan mode [53].

2.7. Statistical Analysis

The data obtained were analyzed using descriptive statistics of continuous variables for normal distribution and homogeneity of variance, using the Kolmogorov–Smirnov and Bartlett test, respectively. Also, analysis of variance was performed using SPSS software version 24 (SPSS Inc., Chicago, IL, USA).

3. Results

3.1. Isolation and Identification of Native Bacterial Colonies from Soils

The bacterial colonies isolated from the soils with the ability to degrade the organochlorine pesticides (OCB) were Gram-positive rods, which were sporulated, thick, and compacted (Figure 2A). On the other hand, the isolated bacterial colonies with the ability to degrade the organophosphorus pesticides (OPB) were Gram-positive rods, which were non-sporulated, elongated, and thin (Figure 2B). Both isolates were positive in the catalase and oxidase tests. The bacterial colonies obtained and labeled as OPB matched the characteristics of the Bacillus cereus bacterial strain with 98% similarity. In addition, the bacterial colonies obtained and labeled as OCB could not be identified within their species, since they matched the characteristics of the bacterial strains Bacillus pumilus and Bacillus brevis, with percentages of 78% and 76%, respectively; however, it was possible to identify their genus as the Bacillus sp. The identification of the OPB and OCB colonies from a comparison with the 16S RDP sequence database, using the SeqMatch tool (http://www.ncbi.nlm.nih.gov/blast/ accessed on 19 July 2019), against the OPB and OCB isolates, respectively, thus indicated that the assembled problem sequences had a higher degree of homology with the sequences of the Bacillus cereus and Paenibacillus lautus species, respectively.

The assembled sequence lengths were 1488 bp for Bacillus cereus, and 1478 bp for Paenibacillus lautus [63].

The results of the taxonomic analysis of the assembled problem sequences against the NCBI ref_seq database indicated that they had 99% and 98% similarity in length with 16S ribosomal gene sequences belonging to the B. cereus and P. lautus species in bacterial growth during pesticide biodegradation bioassays (Figure 3 and Figure 4).

3.2. Determination of Bacterial Biomass During Pesticide Biodegradation Process

After identifying the bacterial strains, vials containing M9 minimal medium with a final concentration of 80 mg·L⁻¹ of the pesticide mixture were inoculated with suspensions of both bacterial species. The readings corresponding to the optical density for the determination of biomass were carried out from within 24 h (1 day) up to a total of 12 days (

Table 1. Growth report, for biomass determination, of B. cereus and P. lautus bacterial isolates exposed to OP and OC mixtures, respectively. Optical density (OD) measured at 600 nm.

Incubation Time	Control—Blank OC	Paenibacillus lautus	Control—Blank OP	Bacillus cereus
(Days)	(Abs)	(Abs)	(Abs)	(Abs)
0	0.007 ± 4.1 × 10−4	0.060 ± 4.7 × 10−4	0.008 ± 9.4 × 10−4	0.013 ± 1.2 × 10−3
1	0.005 ± 4.2 × 10−4	0.080 ± 4.1 × 10−4	0.008 ± 1.2 × 10−3	0.025 ± 1.3 × 10−3
2	0.007 ± 4.7 × 10−4	0.090 ± 4.2 × 10−4	0.007 ± 4.7 × 10−4	0.027 ± 1.2 × 10−3
3	0.006 ± 8.6 × 10−4	0.012 ± 8.1 × 10−4	0.007 ± 4.7 × 10−4	0.044 ± 4.7 × 10−4
4	0.005 ± 4.2 × 10−4	0.014 ± 1.2 × 10−3	0.006 ± 4.7 × 10−4	0.051 ± 1.3 × 10−3
5	0.005 ± 4.1 × 10−4	0.026 ± 1.6 × 10−3	0.005 ± 4.7 × 10−4	0.053 ± 4.7 × 10−4
6	0.006 ± 4.1 × 10−4	0.036 ± 1.3 × 10−3	0.006 ± 8.2 × 10−4	0.056 ± 9.4 × 10−4
7	0.007 ± 4.0 × 10−4	0.066 ± 2.4 × 10−3	0.007 ± 8.1 × 10−4	0.057 ± 1.6 × 10−3
8	0.006 ± 4.2 × 10−4	0.093 ± 8.2 × 10−4	0.006 ± 9.4 × 10−4	0.064 ± 1.7 × 10−3
9	0.005 ± 4.1 × 10−4	0.104 ± 1.2 × 10−3	0.005 ± 4.7 × 10−4	0.076 ± 1.6 × 10−3
10	0.004 ± 4.3 × 10−4	0.103 ± 1.3 × 10−3	0.004 ± 8.1 × 10−4	0.082 ± 1.3 × 10−3
11	0.004 ± 4.7 × 10−4	0.096 ± 1.6 × 10−3	0.005 ± 4.7 × 10−4	0.088 ± 2.1 × 10−3
12	0.005 ± 4.3 × 10−4	0.098 ± 2.0 × 10−3	0.004 ± 4,6 × 10−4	0.091 ± 1.2 × 10−3

), considering that the classical method of quantification of microorganisms is based on the measurement of absorbance at a wavelength of 600 nm of microbial suspension. Figure 4 and Figure 5 show the growth plots of the bacterial strains of P. lautus exposed to the mixture of organochlorine pesticides and B. cereus exposed to the mixture of organophosphorus pesticides, respectively.

These compounds became the strains’ only carbon source. These strains showed a relatively ideal behavior in each of their growth phases, indicating a favorable relationship between their growth and feeding phases in the contaminated media. P. lautus (Figure 4) showed a dormancy (adaptation) phase until day 5; then, on day 8, it reached its phase of highest growth, and remained in its phase of highest biological activity (stationary) until day 10, when it finally reached the cell death phase, and it began the process of biofilm formation from day 11 until the last day of reading. B. cereus (Figure 5) showed a very fast latency (adaptation) phase until day 2; then, on day 4, it reached its phase of highest growth (exponential), and remained in its phase of highest biological activity (stationary) until day 7; finally, it reached the phase of cell death on day 9, and began the process of biofilm formation until the last day of reading. The blank samples (control) were a combination of the M9 medium with the pesticide mixtures at a concentration of 80 ppm, both for OCs (Figure 5) and OPs (Figure 6); these presented a decaying optical density graph, from which it was deduced that the incubation conditions could also influence the degradation of the pesticides, and which allowed for corrections to be made in the exact measurement of OD for the bacterial biomass.

The blank (control), that is, the combination of only the minimum medium M9 and the standard mix of the pesticides at a concentration of 80 mg·L⁻¹, had a decaying optical density graph, from which it can be inferred that the incubation conditions could also influence the degradation of the pesticides; however, this result could only be verified by performing the analyses corresponding to the quantification of the pesticides by gas chromatography. From these results, it can be inferred that the medium doped with the standard mixes is a useful source of carbon for the strains studied, since they showed good growth. However, using this methodology, it is not possible to determine the degradative capacity. To study their degradative potential, it is necessary to quantify the concentration versus the days for which the pesticides were exposed to microbial activity.

3.3. Analysis of Quantification of Pesticide Biodegradation by Gas Chromatography

From these results, it can be deduced that the medium contaminated with the pesticide mixture was a source of carbon for the strains studied, which already showed good growth. P. lautus showed a high capacity to degrade the pesticide lindane during its adaptation phase, to 40.8% of its initial concentration; towards its exponential phase, it maintained a higher affinity to degrade it. However, at the end of its stage of higher biological activity, it was able to degrade a greater amount of the pesticide p,p′-DDT, with a total degradation in its concentration of 65.1% (Figure 7). In general, a very good degradation rate was observed for the group of pesticides to which the bacterial colonies were exposed; these colonies were able to degrade the concentration of some of the pesticides to less than half their initial concentration (

Table 2. Percentages obtained from degradation of OC compounds by P. lautus during its growth phases.

	% Degradation
Incubation Time (Days)	Lindane	Metolachlor	Endrin	p,p′-DDT
5	40.8 ± 0.13	24.7 ± 0.23	25.8 ± 0.23	27.1 ± 0.13
8	48.4 ± 0.24	35.2 ± 0.27	32.9 ± 0.27	35.2 ± 0.26
10	60.6 ± 0.29	45.7 ± 0.10	42.9 ± 0.24	41.5 ± 0.22
12	64.0 ± 0.19	60.8 ± 0.11	55.7 ± 0.18	65.1 ± 0.17

).

B. cereus showed a great capacity for degradation of the pesticides chlorpyrifos and coumaphos (Figure 8). At the end of its stage of greater biological activity, it was able to degrade a greater amount of the pesticide chlorpyrifos, with its concentration being reduced to 73.1% of its starting concentration, and finally, in its cell death phase, after the total time period of the bioassay had been completed, it was able to degrade a greater proportion of the pesticide coumaphos, achieving a reduction in concentration of 79.6% (

Table 3. Percentages obtained from degradation of OP compounds by B. cereus during its growth phases.

	% Degradation
Incubation Time (Days)	Malathion	Chlorpyrifos	Coumaphos
2	5.7 ± 0.12	12.0 ± 0.26	26.7 ± 0.02
4	10.4 ± 0.34	27.4 ± 0.18	36.3 ± 0.19
9	35.5 ± 0.29	73.0 ± 0.16	63.1 ± 0.24
12	52.3 ± 0.17	78.7 ± 025	79.5 ± 0.27

). In general, there was an excellent degradation rate for the group of pesticides to which the bacterial colonies were exposed, with the colonies reducing the concentrations of the pesticides by more than half of their starting concentration.

Paenibacillus lautus isolates were exposed to the mixture of organochlorine pesticides after a total of twelve days of bioassays, and showed a moderate capacity to degrade the pesticides lindane, metolachlor, endrin, and p,p′-DDT, which were degraded in proportions of 65.0%, 60.8%, 55.7%, and 65.1%, respectively (Table 2). Each of them had an initial concentration of approximately 80 ppm.

Bacillus cereus colonies were exposed to a mixture of organophosphorus pesticides, and after a total of 12 days of bioassays, they showed a high capacity to degrade the pesticides malathion, chlorpyrifos, and coumaphos, which were degraded in proportions of 52.37%, 78.79%, and 79.55%, respectively (Table 3). Each of these pesticides had an initial concentration of approximately 80 ppm.

4. Discussion

Microbial degradation is an efficient method of removing chemical contamination from soil. The goal is to eliminate these pollutants and create agricultural soils that are suitable for organic crops [20,21,30,60]. The microbial metabolism of native microorganisms can be exploited for their degradation, since bioremediation is an ecological, cost-effective, and quite efficient method, compared to physical and chemical methods. There are several biodegradation techniques based on bacteria’s enzymatic degradation [21]. The removal efficiency of these processes depends on the type of contaminant. The soil’s chemical and physical conditions also play a significant role. Microorganisms, primarily bacteria or fungi, transform pesticides into less complex compounds, such as CO₂, water, oxides, or mineral salts, which can be used as carbon, mineral, and energy sources [43,63].

Some bacteria have gained interest in agricultural research, as they provide benefits such as fixing atmospheric nitrogen, solubilizing phosphate, remediating heavy metals and pesticides, suppressing methane emissions, and helping in carbon sequestration [64,65]. Different investigations have reported that Bacillus strains can degrade some organophosphorus pesticides, such as malathion, triazophos, and profenofos [5,45,46], but this study is the first report of Bacillus species that can degrade coumaphos. Ishag et al. (2016) [5] conducted a study to identify pesticide-biodegrading microorganisms and to characterize the degradation rates of chlorpyrifos, malathion, and dimethoate. Bacillus safensis, Bacillus subtilis subsp. inaquosorum, and Bacillus cereus strains were isolated from pesticide-polluted soil. B safensis was more effective in degrading chlorpyrifos, and B. cereus was more effective in degrading malathion and dimethoate. Anwar et al. (2009) [47] reported that Bacillus pumilus isolates degraded 89% of chlorpyrifos (1000 mg L⁻¹) in 15 days.

In our work, Bacillus cereus showed a great capacity to degrade the pesticide coumaphos, reducing it by 79.5%. It could also reduce chlorpyrifos by 73.1%. In general, the bacterial colonies exhibited a good degradation rate for the group of pesticides to which they were exposed, reducing the pesticides’ concentrations to less than half of their starting concentration. Leskovac et al. (2023) [66] indicate that different types of microorganisms selectively hydrolyze a variety of organophosphorus contaminants. These include species from the genera Arthrobacter, Enterobacter, Burkholderia, Pseudomonas, Serratia, Sphingobium, Bacillus, and Flavobacterium.

There are reports on OP pesticides’ degrading potential for Bacillus cereus strains; among these, we found a study carried out by Muthukumaravel et al. (2024) [48] which reported the efficient degradation of methyl parathion by B. cereus. Kannahi et al. (2018) [49] found that Bacillus subtilis appeared to be more effective than B. cereus in degrading methyl parathion in soils. Liu and collaborators (2011) [50] reported a degradation potential of 78.8% of B. cereus strains for the pesticide chlorpyrifos, subjected to conditions very similar to those of the tests carried out in this study. Reports have identified other species of bacteria as remediators of organochlorine and organophosphorus pesticides. Lara-Moreno et al. (2022) [67] investigated two chlorpyrifos-degrading strains, Bacillus megaterium and Bacillus safensis, isolated from soils, showing their capacity to degrade up to 99.1% and 98.9% of chlorpyrifos in a solution with an initial concentration of 10 mg. L⁻¹ after 60 days.

Shabbir et al. (2018) [68] isolated Pseudomonas aeruginosa, Enterobacter ludwigii, and Enterobacter cloacae from domestic wastewater to test their ability to degrade chlorpyrifos. Abraham et al. (2014) [69] reported the biodegradation of a mixture of both organophosphorus (chlorpyrifos and monocrotophos) and organochlorine (endosulfan) pesticides, directed by a microbial consortium comprising ten organisms. The bacterial consortium was able to completely degrade the pesticides within 24 h of incubation.

On the other hand, there are several investigations on the degradation of organochlorine pesticides using bacteria isolated from agro-industrial residues and agricultural soils. Barragan-Huerta et al. (2007) [58] proposed a model for the biodegradation of DDT by bacteria grown in micro-niches created in the porous structure of coffee beans, from which five bacterial species were identified: Pseudomonas aeruginosa, P. putida, Stenotrophomonas maltophilia, Flavimonas oryzihabitans, and Morganella morganii. In the degradation of the selected OC compounds, P. aeruginosa and F. oryzihabitans showed better results. Chen et al. (2023) [59] showed the degradation of β-Hexachlorocyclohexanes (β-HCH) in soils using Ochrobactrum sp. and Microbacterium oxydans sp. strains. The degradation bacteria mixed in a ratio of 1:1 had the highest degradation rate for β-HCH, which was 69.57%.

In our study, Paenibacillus lautus showed a moderate capacity to degrade lindane, metolachlor, endrin, and p,p′-DDT, in proportions of 65.04%, 60.80%, 55.74%, and 65.14%, respectively. To date, there are no reports on the possible degradation of OC pesticides by strains of Paenibacillus lautus. Among the reports found, homologous species such as Paenibacillus polymyxa have been found to degrade up to 66.7% of the pesticide p,p′-DDT [70].

5. Conclusions

In this study, organophosphorus and organochlorine pesticide-tolerant bacteria from agricultural soils were isolated, purified, and identified through biochemical and molecular biology tests. The blast analysis showed the identification of the bacterial strains by 16s rDNA sequencing as Bacillus cereus, and Paenibacillus lautus. B. cereus showed a higher degradation potential for coumaphos than chlorpyrifos and malathion. Paenibacillus lautus degraded the pesticides lindane, metolachlor, endrin, and p,p′-DDT in proportions of 65.0%, 60.8%, 55.7%, and 65.1%, respectively. It has been highlighted that P. lautus is a species for which no reports of degradation of OCPs have been found. This implies that this study makes a significant contribution to the scientific community, with a new report for this species.