INTRODUCTION

Organophosphorus chemicals (OF) were developed during the decade of the fifties and used as “nerve gases” during World War II (de Silva et al., 2006).Currently, they are recognized primarily their usage in agriculture as fertilizers and insecticides, existing in the market about fifty thousand kinds of these compounds (Eddleston et al., 2012; Zhang et al., 2014). Because of the indiscriminate and irresponsible usage OF in agro-industry, there is a high rate of acute poisoning characterized by the development of cholinergic syndrome and multiple chronic complications, killing at least 400,000 people annually worldwide (Eddleston & Phillips, 2004; Eddleston et al., 2012).

Colombia ranks third in Latin America in the usage of pesticides after Brazil and Mexico. According to the Food and Agricultural Organization (FAO), the Colom- bian agro-industry constitutes 40% of the workforce and accounts for 50% of national currencies (Bruinsma & FAO, 2003). So, for example, in 2002, the usage of standard agrochemicals like pesticides or fertilizers exceeded 28 million kilograms of which 97% were insecticides (mainly organophosphates and carbamates) followed by herbicides and fungicides (Alegrett, 2002; Mojica & Guerrero, 2010). One of the problems associated with the use of OF is that given their effectiveness, abused products and do not have a proper management othose chemicals or their residues, causing a public health problem (MAVDT, 2006; Murcia & Stashenko, 2008). In 2008 and 2009 the Regional Autonomous Corporation of Southern Bolivar (CSB) conducted a monitoring of the coastal zone of the Caribbean region, finding pesticide compounds in coastal waters and sediments above the detection limit¹ (CBS, 2009). A serious case because of the magnitude of the environmental damage was documented by the Group of Epidemiology and Population Health at the University of Valle, Colombia, in which, in a town of Cartagena de Indias (Ciudadela 2000), pesticides were found buried on the right bank of Troncal de Occidente, affecting four districts and a high school² (MAVDT, 2006).

Given the impact of contamination OF in our country, the usage of techniques that mitigate the impact of these pollutants in soils and water sources is required (Das & Adhya, 2015) O-diethyl O-(3,5,6-trichloro-2-pyridinol. One of the techniques used to reduce environmental OF, is bacterial bioremediation (Abraham et al., 2014). This uses microorganisms capable of degrading complex chemical compounds through various metabolic pathways under aerobic or anaerobic conditions (Cycoń et al., 2009; Marín & Jaramillo, 2015). Which leads to obtain high rates of efficiency and effectiveness at low cost in reducing the level of contamination OF.

In Cartagena of Indias, a qualitative confirmation of OF compounds presence was conducted in contaminated soils in neighborhood Ciudadela 2000 neighborhood and native bacterial strains were isolated. Subsequently, those microorganisms selected in M9 (highly selective medium) with OF as sole carbon source, were characterized bio- chemical and molecularly.

METHODS

Soil samples were collected in plot of Santa Elena land, located in the road out of the municipality of Cartagena to Turbaco, on the right bank of Troncal de Occidente, Cartagena, Bolivar, Colombia. Sampling was conducted according to the methodology proposed by Hudson (1982), which recommends a zig-zag sampling at a depth of 20 cm, soil mix was storage in clean and thick bags for transportation. Collected samples were taken to the laboratory and stored at 4° C in freezer.

Extraction. The extraction of pesticides from soil samples was done using Soxhlet method (Klute y Page, 1982). For this step took 5 to 10 g sample was taken, anhydrous sodium (Na2SO4) sulfate was added up to a complete drying of the mixture. Subsequently, the prepared was placed in an extraction cellulose cartridge with an acetone- hexane solution (1:4) as solvent, for 20 hours. Finally, the sample was concentrated in a rotary evaporator to a final volume of 5 mL and then, with N2 stream to a volume of 1 mL.

Chromatographic analysis. This was carried out according to the methodology used by Marín y Jaramillo (2015). The soil extracts obtained were injected (1µL) in the injection port of a gas chromatograph coupled to a flame ionization detector (GC-FID).

Microbiological treatment. A portion of the collected soil samples was used to achieve expression degrader OF metabolism, according to Cycoń et al. (2009). Monocrotophos first dilution was made to 200 ppm, of this dilution 20 mL were taken and 300 g of soil collected were impregnated, let get dry and placed in dark at 30° C for 30 days. 300 g of soil treated with monocrotophos were sieved through a 2 mm mesh. Then, 1 g of the sieved was added to 9 mL of sterile distilled water. This dilution was the basis for successive solutions from 10-1 to 10-4 using M9 medium as solvent with monocrotophos as sole carbon source (Fernández et al., 2005).

The M9 minimal medium solution (containing 200 ppm monocrotophos) was prepared with hydrated sodium acid phosphate (6 g), potassium acid phosphate (3 g), ammonium chloride (4 g), sodium chloride (0.5 g), manganese sulfate heptahydrate (0.25 g), hydrated calcium chloride (0,0168 g), monocrotophos pattern (0.2g) and it was completed to 1L with distilled water and constant stirring (Elbing y Brent, 2002). The pH was adjusted to 7-7.2 with 1N hydrochloric acid and 5N sodium hydroxide and refrigerated until its usage. 16 solid phase media were prepared from M9 min- imal medium solution by using agar-agar according to the manufacturer´s instructions.

For isolation of bacterial microorganisms growing on M9 minimal medium, massive seeding in soil diluted in nutrient agar was made. Then it was incubated for 1 week at 36° C ± 1. Once colonies grew, the largest ones and those with better organoleptic properties were inoculated into M9 liquid medium. One week after seeding the colonies in the liquid matrix of M9 medium, bacterial growth was monitored by UV Visible spectrophotometry (UV/VIS) and consecutive readings were made during four days. For identification, isolated bacterial colonies on solid M9 medium were taken and replicated three times on nutrient agar by using the exhaustion technique and thus ensure the isolation of each. After the third replication, colonies were subjected to Gram staining and seeded on bacterial identifica- tion kits by biochemical methods BBL Crystal ©.

Isolated colonies, purified and biochemically characterized, were genotyped by 16S ribosomal gene amplification through polymerase chain reaction (PCR) and sequencing of a 1465 pb fragment. Once fragment sequences were obtained they were assembled and compared to sequences deposited in the databases NBCI (National Center for Biotechnology Information) and RDP (Ribsomal Database Project). Commercially procedures performed by Corpogen (Bogotá-Colombia).

Validation of the OF pesticide degradation by bacterial action. Degrading activity verification of isolated and identified bacteria was performed by measuring the OF concentration by using GC/FID. The bacterial strains were seeded in the liquid matrix M9 using Eppendorf © tubes and they were incubated at 36 ± 1° C for 7 days. The initial value of concentration of M9 with Monocrotophos to 200 ppm was taken as reference and 24 hours later the first measurement of concentration with the chromatograph was made, with two subsequent measurements every 24 hours for 3 days.

Statistic 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 (Allen, 1976), respectively.

RESULTS AND DISCUSSION

The soils analyzed showed the presence of pesticides OF. Three bacterial strains with acceptable rates of growth and ability to survive in saturated media with OF (pattern OF Monocrotophos), were obtained (Table 1).Once isolated and purified the strains present in the M9 minimal medium were identified as Gram negative bacilli not sporulated suggestive of Enterobacteriaceae. After confirming the acceptable growth of bacteria in the presence of an OF pattern as sole carbon source, both biochemical and molecular identification were performed to determine genus and species (Table 2).

TABLE 1

Bacterial growth of the strains under study

* The data are presented as optical density ± standard error.

TABLE 2

Biochemical behavior of isolate strains obtained by BBL Crystal©kit

Biochemical characterization of C1, C2 and C3 strains showed the presence of three different species: Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa, re- spectively. However, the results of sequencing the 16S ribosomal gene, located C1 and C2 strains relative to Enterobacter genus, which differs from that found with Crystal® BBL identification techniques. By comparing these strains similarities were found in colony morphology, microscopic characters, not oxidative metabolism, being not undemanding facultative anaerobic and having similar metabolism. Even taxonomically the two species are part of the Enterobacteriaceae family. It was found that between the C1 and C2 Strains there was genetic similarity within 99 and 100% with Enterobacter cancerogenus and other unidentified species, possibly, because a single gene and not the entire genome of the species is being used.

The 16S gene sequence analysis showed for strain 1, a sequence of 1475 bp that relates to 99% with Enterobacter species, among which are Enterobacter cancerogenus and other unidenti- fied. However, the classifier RDP (Ribosomal Database Project) only arose to an unclassified sequence of Enterobacteriaceae group (Figure 1). As for strain 2, the results of taxonomic analysis indicate that the sequence 1477 pb, has a 99% identity in 100% of its length, with sequences belonging to several species of Enterobacter genus and other unidentified, being nearer to Enterobacter cancerogenus (Figure 1). Finally, strain 3 had a 99% of identity with several species of Pseudomonas, being genetically closer to a sequence of the Pseudomonas aeruginosa species (Figure 1).

The liquid culture media were analyzed by GC/FID to determinate present of organophosphorus pesticide. Besides of the three strains tested a white or negative control was done, in which the medium was used unseeded. The information obtained from the GC/FID analysis, which monitored the degradation of monocrotophos standard by the three bacterial isolates (C1, C2 and C3) and the negative control is shown in Table 3. After 72 hours in incubation was evidenced a marked decreased pesticide, degradation monocrotophos standard was up to 100% after 48 hours of incubation (Figure 2).

The degrading power of the soil isolated strains was demonstrated by finding optical densities near 0.7, in sim- ilar research, the optical densities found after one or more weeks of growth were 0.7 to 10 (Cycoń, et al., 2009; (Cycoń et al., 2009, 2013; Acuña et al., 2010). Even in bacterial strains of the same taxonomic family used in this research (Cycoń et al., 2009, 2013). The differences found between the two methods of identification can be attributed to genetic variability due to exchange of the material between species (Winn & Koneman, 2006).

The degrading power of the soil isolated strains was checked through degradation monocrotophos. Apparently, this process produces secondary metabolites, although they are also considered pollutants. Probably, these are degraded by the same metabolic pathway, to achieve harmless compounds. The Monocrotophos biotransformation develops through three different metabolic reactions: N-demethylation, Odemethylation, and a bond cleavage of vinyl phosphate. Generally, monocrotophos is metabolized in Nhydroxy-methylamide (Rao, 2006) and n-me- tilacetoamida. The latter tends to accumulate in contaminated soils (Gundi & Reddy, 2006). Monocrotophos is converted into hydroxymethyl using oxidative metabolic pathway (Hodgson, 2012). Metabolites produced by the bacteria isolated from soil could be generating inorganic phosphate and bacterial enzymes such as phosphatase, which enhance its ability to capture such molecules in phosphate free media, allowing the growth of the colonies (Ríos & Solari, 2010).

FIGURE 1
Tree of distances constructed from 50 best arrangements regarding the NCBI database. The nodes (•) corresponding to the query sequence belongingto clade Enterobacter cancerogenus / Enterobacter hormaechei (Strain 1 and 2) and Pseudomonas aeruginosa (strain 3) are showed in the box.

TABLE 3

Percentage reduction of monocrotophos standard by the isolates and the white reaction

Regarding the rate of decomposition and disappearance of organophosphates, carbamates and other compounds, it is only relative, since some factors such as the chemical structure of the compound, soil type, organic matter content, content and nature of clay minerals in the soil, granulometric composition, pH, moisture and temperature can influence decisively in the degree of persistence (Bhadbhade et al., 2002). Pierre & Betan- court (2007) showed that OF pesticides used in agriculture generate bioaccumulation and can remain as contaminants for a long period of time (Abo-El-Seoud et al., 1995; Pierre & Betancourt, 2007).

FIGURE 2
Percentage reduction of monocrotophos standard by the strains under study (C1, C2, C3).

CONCLUSION

• Three bacterial strains (C1, C2, C3) were isolated from soils contaminated by organophosphate pesticides. According to the study at the molecular level of gene ribosomal 16S, it was determined that cultured bacteria from C1, C2 and C3 samples belong to the Enterobacteriaceae family. C1 and C2 had a genetic similarity between 99 and 100% with Enterobacter cancerogenus and other un- specified species. However, closer interaction with each of them is quite different, so it can be concluded that they may be subspecies.

• Cultured bacteria of sample C3 were located within the Enterobacteriaceae family with a degree of similarity of 99 to 100% with the species Pseudomonas aeruginosa. These species demonstrated their ability to degrade pesticides OF.

Acknowledgements

This work was funded by the University of Cartagena, with strong support from the Research Vice-Rectory. We thank Agrochemical Research Group of the Faculty of Nat- ural Sciences. To Orlando de la Rosa for his collaboration in chromatographic analysis. To chemists Hector Pertuz A, and Fernando Fernandez.

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Notes

Información adicional

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