(234z) Evaluation of Microorganism Resistant to Heavy Metal Isolated from Contaminated Water and Soil Used in the Agriculture Sector in Ecuador

This research focuses on the evaluation of microorganism taken from a water canal used for irrigation, soil crop production of vegetables and fruits in Latacunga, Ecuador. This region supports a huge demand of fruits and vegetables. However, some of the water and soil used is contaminated by heavy metal. There is a need to find environmentally friendly techniques that can increase the production and quality of crops in order to meet growing water requirements from municipalities, industry and agriculture. The evaluation and identification of microorganism will be the step to recover polluted zones and search for better remediation techniques. Ecuador is a country located in South America; it has an area of 283 561km². The population of Ecuador is around 16 millions of habitants. The advantages of the geographical location of Ecuador are its agricultural and agro industrial production. This country is ideal for growing a wide of variety of fruits and vegetables. Several rivers, snowy mountains and paramos surround Latacunga. This province is recognized because of its industrial sector, tanneries, car washer and shoes factories.

Farmers require water sources for agriculture, which are distributed by canals. However, water sources can be contaminated due to human activities and industrial activities. The aim of this study is to evaluate the resistance of bacteria to heavy metal by isolating, purifying and transferring microorganisms present in the water of Rio Cutuchi. Due to the use of contaminated water from canal for crops and farming, there is a need to develop further water analyses. There are different physicochemical and remediation techniques to degrade contaminants. Bioremediation is an action and process taken place in order to bio transform an environment using microorganism or microbial processes to degrade pollutants. Microorganisms can detoxify metals by valence transformation, extracellular chemical precipitation, or volatilization. They can enzymatically reduce some metals in metabolic processes that are not related to metal assimilation. Therefore, it is necessary to analyze microorganisms, which resist heavy metals, since this is a technique that will be environmentally friendly, but still has some drawbacks in the manipulation and control of microorganism. Bioremediation research performed in Ecuador has been mainly on petroleum remediation. Since polluted water for farming use has not been investigated, this study brings options to identify microorganism that resist heavy metals. Future work scopes propose techniques of remediation, which interest farmers, municipalities industry and agriculture sectors.

The project consisted on collecting water and soil samples contaminated by heavy metals from different locations in the canal Latacunga â?? Salcedo - Ambato. Selective media was prepared to isolate bacteria and fungi microorganism, incubation was done at 30 ^oC for 48 hours. Microorganism were purified and transferred. Forty bacteria were selected for resistance studies to heavy metals and microorganism characterization. Biochemical tests and macroscopic examination had been applied to the strains, the results indicate that bacteria are identified as gram negative and refer to coccus bacteria.

Microorganisms resistant to the following heavy metal were evaluated: Mercury (Hg), Chromium (Cr), Cooper (Cu), Zinc (Zn), Niquel (Ni), and Cadmium (Cd). The resistance of bacteria was analyzed at a solid and liquid phase, at a concentration of 2mM of heavy metal. Reactants used to establish the concentration were mercury (II) oxide (HgO), Chromium (III) oxide (Cr₂O₃), Copper (II) sulfate (CuSO₄),Zinc chloride (ZnCl₂), Nickel (II) Sulfate (NiSO₄), Cadmium chloride (CdCl₂). For the evaluation of resistance of heavy metal in the liquid phase, nutrient broth was prepared and bacteria was incubated for 24 hours at 30^oC. To determine bacteria growth, spectrophotometry was used and the absorbance was measured at a specific wavelength. Results indicate that 80 % of the strains were resistant to Mercury (Hg), 40% were resistant to Chromium (Cr), 22 % were resistant to Cupper (Cu), 72% were resistant to Zinc (Zn), 2% were resistant to Niquel (Ni) and 30% were resistant to Cadmium (Cd).

When analyzing the strains in the solid phase, Difco^TM Potato Dextrose Agar was prepared and the growth of microorganism was evaluated at 24 hours, 48 hours and 72 hours. A total of 40 strains were analyzed at each time and growth was evaluated. In this study, 82% resisted Mercury (Hg), 80% resisted Chromium (Cr), 55% resisted Cupper (Cu), 80% resisted Zinc (Zn), 20% resisted Niquel (Ni) and 47% resisted Cadmium (Cd). The values obtained for the resistance of heavy metals for the liquid phase shows that microorganism isolated from the canal resist most to Mercury (Hg) and Zinc (Zn), in case of mercury the growth of bacteria is 99.4% respect to the control in which heavy metal is not inoculate. The same results are shown in the solid phase. The heavy metal that was less resistant was niquel (Ni) in which, different strains results in the liquid and solid tests. This result shows that there are potential uses of bacteria to remediate contamination with Mercury (Hg), Zinc (Zn) and Chromium (Cr).

Future work consists on counting bacteria by the method of massive stamping drop plate, and the identification of bacteria applying molecular techniques, polymerase chain reaction (PCR). By identifying the microorganism that can resist higher concentration of heavy metal and can decontaminate zones with heavy metals, this results can benefit future work related to establishing microorganism with bioremediation potential for zones were the use of water for farming is not well controlled and treated.