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Biorremediaci�n de suelos contaminados con hidrocarburos mediante microorganismos nativos

 

Bioremediation of soils contaminated with hydrocarbons through native microorganisms

 

Biorremedia��o de solos contaminados com hidrocarbonetos atrav�s de microrganismos nativos

 

 

 

 

 

 

 

 

 

 

 

 

Correspondencia: : jose.agreda2101@utc.edu.ec

 

Ciencias T�cnicas y Aplicadas

Art�culo de Investigaci�n

 

* Recibido: 15 de mayo de 2024 *Aceptado: 20 de junio de 2024 * Publicado: �31 de julio de 2024

 

        I.            CAREN, Universidad T�cnica de Cotopaxi, Latacunga, Ecuador.

      II.            CAREN, Universidad T�cnica de Cotopaxi, Latacunga, Ecuador.

   III.            CAREN, Universidad T�cnica de Cotopaxi, Latacunga, Ecuador.

   IV.            CIYA, Universidad T�cnica de Cotopaxi, Ecuador.

Resumen

El presente trabajo de investigaci�n estuvo dirigido al an�lisis de microorganismos presentes en los suelos que han sufrido derrames de petr�leo en las cercan�as de las estaciones productoras de petr�leo, ubicadas en la zona de Dayuma, provincia de Orellana en el campo petrolero Auca, con el objetivo de evaluar la capacidad de remediaci�n de suelos de dichos organismos unicelulares, que han sido expuestos a hidrocarburos, debido a accidentes operacionales en la zona; el prop�sito de este estudio fue verificar la presencia y el desempe�o de la microbiota resistente a los carburos, observando su desempe�o de remediaci�n con est�mulos naturales de atenuaci�n, tales como; m�todos redox y adici�n de glucosa para comparar el potencial de los microorganismos en la remoci�n de sus contaminantes en el suelo. Se realizaron t�cnicas de aislamiento de microorganismos cultivables, mediante el uso de medios de cultivo espec�ficos luego de realizar diluciones seriadas hasta 10-5. Posteriormente, se clasificaron los grupos microbianos asociados, a trav�s de diferentes m�todos, como morfol�gicos, tinci�n de Gram y pruebas bioqu�micas, con el fin de identificar el g�nero bacteriano de los microorganismos. Adem�s, se implement� un dise�o de bloques al azar (DBA) con tres tratamientos TO (Suelo + Hidrocarburo + agua), T1 (Suelo + Hidrocarburo + agua + aireaci�n) y T2 (suelo + hidrocarburo + agua + melaza). En el an�lisis estad�stico de los resultados se utiliz� un ANOVA con una prueba de Tukey al 5%. Obteniendo como resultados, la presencia de bacterias pertenecientes a la familia Enterobacteriaceae, siendo el g�nero predominante Serratia Sp con un porcentaje de 39%. Otros g�neros identificados fueron Shigella Sp con un 17%, Hafnia Sp con un 11%, Yersina Sp con un 11%, Enterocolitica Sp con un 6%, Citrobacter Sp Freundi Sp con un 6%, Proteus Sp con un 6%, Klebsiella Sp con un 6% y Enterobacter Sp con un 6%. Finalmente, se observ� que de los tres tratamientos aplicados se logr� degradar los hidrocarburos presentes en el suelo contaminado, siendo el tratamiento 3 el m�s efectivo con una tasa de degradaci�n de hidrocarburos totales (TPHs) de 7484 mg/kg. Concluyendo que, si bien existen microgramos capaces de resistir los efectos de los hidrocarburos presentes en el suelo de la parroquia Dayuma, tambi�n son capaces de degradar en un 77% en suelos contaminados por hidrocarburos.

Palabras Clave: Diluciones; Medios de cultivo; Colonias bacterianas; TPHs; Metabolismo.

 

 

 

Abstract

This research was directed to the microorganisms analysis present in the soils, what have suffered oil spills near the petroleum production stations, located at the Dayuma�s area, Orellana province in the Auca oil field with the aim by assessing the soils remediation capacity of said unicellular organisms, which have been exposed to hydrocarbons, due to operational accidents in the area; the purpose this study was to verify the carbide resistant microbiota presence and performance, looking at its remediation performance with natural attenuation stimuli, such as; redox methods and glucosa addition to compare the microorganisms potential their contaminants removal in the soil. It was carried out cultivable microorganisms isolation techniques, through specific culture media use after making serial dilutions up 10-5. Subsequently, they were classified the associated microbial groups, through different methods, such as morphological, Gram staining and biochemical tests, the purpose was to identify the microorganisms bacterial genus. Further, it was implemented a randomized block design (DBA) with three treatments TO (Soil + Hydrocarbon + water), T1 (Soil + hydrocarbon + water + aeration) and T2 (soil + hydrocarbon + water + molasses). In the results statistical analysis, it was used an ANOVA with a 5% Tukey test. Getting as results, the bacteria presence belonging to the Enterobacteriaceae family, by being the predominant genus Serratia Sp with a 39% percentage. Other identified genera included Shigella Sp at 17%, Hafnia Sp at 11%, Yersina Sp at 11%, Enterocolitica Sp at 6%, Citrobacter Sp Freundi Sp at 6%, Proteus Sp at 6%, Klebsiella Sp with 6% and Enterobacter Sp with 6%. Finally, it was observed, which applied two from three treatments achieved to degrade the hydrocarbons present in the contaminated soil, with the treatment 3 was the most effective with a total hydrocarbons (TPHs) 7484 mg/kg degradation rate. Concluding that, if there are micrograms capable by resisting the hydrocarbons effects present in the soil from Dayuma parish, they also are capable by degrading 77% in soils contaminated by hydrocarbons.

Keywords: Dilutions; Culture media; Bacterial colonies; TPHs; Metabolism.

 

Resumo

Esta pesquisa foi direcionada para a an�lise dos microrganismos presentes nos solos, que sofreram derrames de petr�leo junto �s esta��es de produ��o de petr�leo, localizadas na zona de Dayuma, prov�ncia de Orellana, no campo petrol�fero de Auca, com o objetivo de avaliar a capacidade de remedia��o dos solos dos referidos organismos unicelulares, que foram expostos a hidrocarbonetos, devido a acidentes operacionais na �rea; o objetivo deste estudo foi verificar a presen�a e o desempenho da microbiota resistente ao carboneto, observando o seu desempenho na remedia��o com est�mulos de atenua��o natural, tais como; m�todos redox e adi��o de glicose para comparar o potencial de remo��o de contaminantes dos microrganismos no solo. Foram realizadas t�cnicas de isolamento de microrganismos cultiv�veis, atrav�s da utiliza��o de meios de cultura espec�ficos ap�s dilui��es seriadas at� 10-5. Posteriormente, foram classificados os grupos microbianos associados, atrav�s de diferentes m�todos, como morfol�gicos, colora��o de Gram e testes bioqu�micos, o objetivo foi identificar o g�nero bacteriano dos microrganismos. Al�m disso, foi implementado um delineamento em blocos casualizados (DBA) com tr�s tratamentos TO (Solo + Hidrocarboneto + �gua), T1 (Solo + hidrocarboneto + �gua + aera��o) e T2 (solo + hidrocarboneto + �gua + mela�o). Na an�lise estat�stica dos resultados foi utilizada a ANOVA com teste de Tukey a 5%. Obtendo como resultados a presen�a de bact�rias pertencentes � fam�lia Enterobacteriaceae, sendo o g�nero predominante Serratia Sp com uma percentagem de 39%. Outros g�neros identificados inclu�ram Shigella Sp com 17%, Hafnia Sp com 11%, Yersina Sp com 11%, Enterocolitica Sp com 6%, Citrobacter Sp Freundi Sp com 6%, Proteus Sp com 6%, Klebsiella Sp com 6% e Enterobacter Sp . Por fim, observou-se que aplicou dois dos tr�s tratamentos conseguidos para degradar os hidrocarbonetos presentes no solo contaminado, sendo que o tratamento 3 foi o mais eficaz com uma taxa de degrada��o de hidrocarbonetos totais (TPHs) de 7484 mg/kg. Concluindo que, se existem microgramas capazes de resistir aos efeitos dos hidrocarbonetos presentes no solo da freguesia de Dayuma, tamb�m s�o capazes de degradar 77% em solos contaminados por hidrocarbonetos.

Palavras-chave: Dilui��es; Meios de cultura; Col�nias bacterianas; TPHs; Metabolismo.

 

Introduction

Soil contamination with hydrocarbons is a major environmental problem that affects numerous ecosystems worldwide (1). These toxic compounds, derived from industrial activities and accidental spills, pose a serious threat to soil quality and, consequently, to human health and biodiversity (2). Considering this issue, bioremediation has emerged as a promising environmental restoration strategy, utilizing native microorganisms that can naturally degrade and eliminate petroleum contaminants from the soil (3).

 

This study focuses on the bioremediation of hydrocarbon-contaminated soils through native microorganisms at the laboratory level (5). The central objective of this research is to evaluate the remediation capacity of soil that has been previously treated for hydrocarbon contamination, through the application of physical and microbiological treatments, in order to determine their effectiveness in restoring soil properties and health (6).

This investigation aimed to contribute to the advancement of bioremediation technology and the understanding of soil recovery processes affected by hydrocarbons, providing crucial information for environmental management and the protection of our surroundings (7). Throughout this study, we will explore the microbiological strategies employed in the laboratory to rehabilitate contaminated soils, as well as their viability in the effective remediation of these degraded ecosystems (8).

 

Methodology

Soil Sampling

To collect representative samples of contaminated soil, the systematic random method was used, ensuring an impartial and uniform selection of sampling points (9). A 10 m� plot was established, and sampling points were randomly distributed within this area. Then, a 30 cm deep excavation was carried out at each selected point (10).

For soil samples were collected, each weighing 5 kg, and placed in polyethylene bags with a pore diameter of 0.8 �m to prevent cross-contamination. Subsequently, they were mixed homogeneously to obtain four subsamples of 1 kg each (11). These subsamples were placed in properly labeled Ziploc bags with detailed information such as the collection site, date, sample type, responsible person, and corresponding sample number.

 

Control of Culture Media

In order to evaluate the proper functioning of the culture media used, a control of culture media was carried out. For this, thioglycolate medium, an enriched agar that facilitates the development of various microorganisms (12), was used. Additionally, MacConkey agar, a selective differential medium that inhibits the growth of Gram-positive bacteria and allows the distinction of Gram-negative bacteria, was used. In both culture media, an Enterococcus strain was inoculated to obtain a positive quality control and evaluate its appropriate growth.

Similarly, to establish a negative control, the same culture media were used without inoculating any microorganisms; this was done to ensure that the culture media were not contaminated at the time of seeding (13).

 

Serial Dilutions

For the isolation of native microorganisms, the serial dilution technique was used, which was also used to quantify the microorganisms on a culture plate. The soil sample was diluted 1 in 10. That is, 25g of soil in 125 ml of peptone water (10^-1), 1g of the previous solution in 9 mL of peptone water (10^-2), and so on until 10^-5.

Dilutions of 10^-2, 10^-3, and 10^-4 were plated in duplicate, with 0.1 mL (100 �L) of each dilution being taken with a micropipette and plated on petri dishes with thioglycolate agar by spread plate method. Finally, they were incubated at 37 �2 �C for 48 hours under optimal conditions for bacterial growth.

 

Quantification

To determine the number of Colony Forming Units per gram (CFU/g) in each of the samples, the number of colonies present in the countable dilution (10^-3) was counted. It was considered that one colony was equivalent to one CFU/g. This value represented the amount of CFU/g in the volume of the drop that was deposited by the replicator. Once the number of colonies in 1 ml (100 �L) was determined, the result was multiplied by the dilution factor used and divided by the volume in ml of the sample plated. In this way, the concentration of CFU in 1 ml (100 �L) of the sample was calculated

 

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Macroscopic Characterization 

In the study of macroscopic characterization, the method proposed by MacFaddin in 2006 was employed (14). During this stage, the colonies formed on the surface of the culture medium were examined, analyzing both the top and bottom of the Petri dish. The objective was to identify the macroscopic characteristics of the colonies and distinguish those that are common and specific to each bacterial group. Through this approach, different morphotypes were identified and classified based on their macroscopic characteristics. Consequently, the following criteria were considered:

 

Axenic Bacterial Isolation 

After obtaining a colony count result from the culture media, a new culture medium was inoculated to obtain axenic or pure cultures of the morphologically different colonies found. The isolation was carried out in thioglycolate medium, and a sterilized bacteriological loop was used to obtain a sample of the colonies and inoculate it using the streaking method. The plates with pure growth were placed in the incubator at a constant temperature of 37 �2 �C for a period of 24 to 48 hours (15). Once the required time had elapsed, the plates were used for characterization, identification, and cryopreservation.

 

Microscopic Characterization (Gram Staining) 

Gram staining is an effective and rapid technique that allowed us to distinguish between two categories of bacteria: Gram-positive and Gram-negative bacteria (16). The Gram staining technique is based on the structural differences of bacteria, such that Gram-positive bacteria acquire a purple color in Gram staining because the dye is retained in the dense peptidoglycan layer surrounding the cell, while Gram-negative bacteria do not retain the crystal violet during Gram staining due to their thinner peptidoglycan layer and therefore take on the color of the final counterstain safranin, appearing red or pink (17).

 

Gram Staining 

For microscopic characterization, the following criteria were considered: Shape, Grouping, and Gram Staining (18).

 

Figure1: microscopic morphology of bacteria

 

Biochemical Determination of Bacteria

 In biochemical tests, various specific culture media were used to evaluate different metabolic aspects of microorganisms. These media included MacConkey Agar for the detection of lactose-fermenting gram-negative bacteria, Lysine Iron Agar to investigate the ability of some bacteria to break down lysine and produce sulfur reactions, triple sugar iron (TSI) Agar to analyze sugar fermentation and hydrogen sulfide production, SIM Agar to evaluate sulfur production, indole, and bacterial motility, Citrate Agar to determine the ability of bacteria to use citrate as the sole carbon source, and Urea Agar to verify urease enzyme activity(19).

 

Preparation of Cryopreservation Tubes with Glycerol and BHI Medium 

An aqueous solution with glycerol preservative was developed to preserve bacteria in the logarithmic growth phase (24 hours of incubation) from the axenic medium (20).

 

Inoculation of Bacteria 

The specific colony was identified and transferred to Eppendorf tubes using a previously sterilized metal loop. The tubes were subjected to vigorous shaking for 15 seconds in the vortex to ensure homogeneous distribution of the bacteria in the medium (21).

 

Storage of Prepared Tubes 

Finally, the Eppendorf tubes were placed in a freezer at -20�C, ensuring future research.

Table 1: Solution to preserve isolated bacteria

 

 

Results and discussion

Three treatments were carried out. Treatment 1 was considered the control group, in which a soil sample containing water and hydrocarbons was prepared. In Treatment 2, oxidation-reduction (redox) was applied, which is considered a passive volatilization technique for volatile contaminants. According to Manrique & Guisella (22), this method consists of promoting volatilization through periodic soil removal. In this case, soil removal was performed every 2 days for a month to promote the release of hydrocarbons present in volatile form. Subsequently, a TPHs test was conducted to evaluate the contamination levels in the treated soil.

In Treatment 3, molasses addition was applied, which is based on promoting the growth of microorganisms with the ability to decompose contaminating substances (bioremediation process). This can involve optimizing soil conditions to enhance the effectiveness of existing microorganisms or introducing new species. To promote biotic actions, specific soil conditions can be improved, such as adding nutrients, water, oxygen, and adjusting the pH (23). In this treatment, water with molasses was introduced to provide additional nutrients to the microorganisms present in the hydrocarbon-contaminated soil. Subsequently, TPHs tests were conducted to evaluate the degree of contamination in the treated soil. Finally, a comparison was made with the results obtained in the previous treatments to determine the effectiveness of the microbiological methodology in remediating contaminated soil.

Table 2 provides detailed information on the initial and final values of TPHs. In the initial phase, TPH levels were at 33,026 mg/kg, and by the end of the experiment, treatment 3 recorded a notable reduction, with 7,484 mg/kg of TPHs degraded. This decrease represents a 77% reduction in the total TPH content. This change is attributed to the positive influence of bacterial metabolites and the addition of extra nutrients, such as molasses, which played a crucial role in the hydrocarbon degradation process. In this context, the Enterobacteriaceae bacterial consortium, by multiplying more rapidly, significantly contributed to accelerating the decomposition of TPHs (24).

Table 2: Total Petroleum Hydrocarbons at the beginning and end of the treatment.

 

Analysis of Variance (ANOVA)

It is observed that the p-value in the ANOVA table is <2e^-16. Since it is less than 0.05, there is statistical evidence to reject Ho, meaning that there are differences in at least one of the treatments. Therefore, the TUKEY test is performed to determine which group means are different.

 

Table 3: Analysis of Variance

 

It is observed that the p-value in the ANOVA table is <2e-16. Since it is less than 0.05, there is statistical evidence to reject the alternative hypothesis, meaning that there are differences in at least one of the treatments. Therefore, the TUKEY test is performed to determine which group means are different (25).

In Figure 2, it is shown that there is a statistically significant difference between the average soil degradation of each treatment at the 0.05 significance level. Concluding that the best treatment is treatment 3, due to the greater hydrocarbon degradation in the soil and the validity of the data

Figure 2: Tukey 5% analysis of the treatments

 

Analysis of Sample Variables

Moisture of the Hydrocarbon-Contaminated Sample

The following table describes the analysis of the moisture of the hydrocarbon-contaminated sample.

 

Table 3: ANOVA and Tukey Analysis at 5% of the Sample Moisture

 

Table 3 summarizes the average moisture levels in various treatments. The data reveals that treatment T1 has an average of 52.22%, closely followed by T2 with 52.92%, while T3 shows a higher average of 60.14%. It is important to emphasize that the recorded moisture levels are within the ideal ranges for the execution of bioremediation procedures. It is important to keep in mind that the optimal conditions for bacterial growth in bioremediation processes are usually between 30% and 90% of ambient moisture (26).

 

Electrical Conductivity of the Hydrocarbon-Contaminated Sample

The following table describes the analysis of the electrical conductivity of the hydrocarbon-contaminated sample.

 

Tabla 4: An�lisis de ANOVA y Tukey al 5% de la conductividad el�ctrica de la muestra.

 

Table 4 analyzes the relationship between treatments and Electrical Conductivity (E.C.), a key measure of soil salinity that can vary due to various factors, such as the presence of hydrocarbons in the soil. In this context, the results indicate that treatments T1, T2, and T3 were evaluated in terms of their E.C. Treatment T1 shows the highest E.C., with a value of 6.75 dS/m, suggesting a higher concentration of saline compounds in the soil. Treatments T2 and T3 have similar E.C. values, both recording 6.37 dS/m. These Electrical Conductivity (E.C.) values indicate the influence of hydrocarbons on soil salinity (27).

 

pH of the Hydrocarbon-Contaminated Sample

The following table describes the analysis of the pH of the hydrocarbon-contaminated sample.

 

Table 5: ANOVA and Tukey Analysis at 5% of the Sample pH

 

Table 5 shows the average pH values for each treatment. The most notable results are those of T2 and T3, which have the highest pH in the process, recording 8.18, while T1 has the lowest pH with 8.16. These differences in pH are due to the addition of redox and molasses in these treatments, which has contributed to maintaining a similar pH among them (28).

 

Conusions

From three samples with 2 duplicates each, a total of 18 bacterial colonies were obtained. These colonies were classified as Gram-negative bacilli using the Gram staining technique. Subsequently, biochemical tests were carried out to characterize and determine the genus of the identified species. The cryopreservation of the bacteria was carried out at -20�C, proving to be a highly effective technique for the long-term preservation of bacterial strains. This ensures their viability and functionality, which is essential for their usefulness in future research.  Treatment 3 achieved a remarkable efficiency by degrading 77% of the contamination present in the soil. These findings highlight the feasibility and potential of the remediation strategies used to mitigate the negative effects of hydrocarbon contamination in the soil. However, it is crucial to continue researching and refining these remediation techniques to further optimize their effectiveness and consider their large-scale application in similar environmental contamination situations.

 

Referencias

      1.            Almaz�n Casta�eda, P. (2023). Fitorremediaci�n de Helianthus annuus L. de un suelo contaminado con un hidrocarburo de petr�leo (gasolina), asistida con bacterias y hongos promotores del crecimiento vegetal. [Tesis Posgrado, Colegio de Postgraduados]. Repositorio Institucional. Obtenido de http://hdl.handle.net/10521/5116

      2.            Ant�nez S�nchez, A. F., & Bruz�n Viltres, C. J. (Diciembre de 2012). LOS CONFLICTOS AMBIENTALES EN CUBA, SOLUCI�N DENTRO DEL DERECHO INTERNO. SU TRATAMIENTO TE�RICO DOCTRINAL EN OTROS SISTEMAS DE DERECHO COMPARADO. SciELO(35), 48. doi:http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S1909-24742012000200016

      3.            Bustamante Velarde, A. (2022). Biorremediaci�n de suelos contaminados con aceite residual de talleres automotrices: Revisi�n sistem�tica, 2022(Tesis de Ingenieria, Universidad Cesar Vallejo). Biorremediaci�n de suelos contaminados con aceite residual de talleres automotrices: Revisi�n sistem�tica, 2022. Repositorio de la Universidad C�sar Vallejo, Lima. Obtenido de https://hdl.handle.net/20.500.12692/104119

      4.            TULSMA. (2017). TEXTO UNIFICADO DE LEGISLACION SECUNDARIA DE MEDIO AMBIENTE. Obtenido de https://www.ambiente.gob.ec/wp-content/uploads/downloads/2018/05/TULSMA.pdf

      5.            Lozano Mahecha, R. A., & L�pez L�pez, K. (Junio de 2022). Aislamiento y caracterizaci�n de bacterias end�micas colombianas con capacidad de degradar tolueno. Revista Colombiana de Biotecnolog�a, 24(1), 13. doi:https://doi.org/10.15446/rev.colomb.biote.v24n1.98613

      6.            Guti�rrez Garc�a, M. (2022). Revisi�n sobre la utilizaci�n de bioindicadores para analizar la calidad del aire en contextos urbanos. [Monografia, Universidad Nacional Abierta y a Distancia]. Repositorio Institucional. Obtenido de https://repository.unad.edu.co/handle/10596/38777

      7.            Lopez Dinorin, R. (2020). Estudio electroosm�tico de la migraci�n de hidrocarburos en suelos finos. [Tesis de Maestr�a, Tecnol�gico Nacional de M�xico]. Repositorio Institucional. Obtenido de http://200.188.131.162:8080/jspui/handle/123456789/385

      8.            Marroquin Sanchez, G. (2007). Formaci�n de sedimentos durante el hidrotratamiento catalitico de crudo pesado [Tesis de Doctorado, Instituo Politenico Nacional]. Repositorio Institucional . Obtenido de http://tesis.ipn.mx/handle/123456789/167

      9.            Acevedo Rojas, N. (2023). Dise�o de una herramienta pedag�gica para la ense�anza de la nomenclatura y propiedades de los hidrocarburos que contribuya al desarrollo del pensamiento creativo. [Tesis de Maestr�a, Universidad Nacional de Colombia]. Repositorio Institucional. Obtenido de https://repositorio.unal.edu.co/handle/unal/83864

  10.            Bacterias pat�genas presentes en dos zonas del Rio Apatlaco, en el Estado de Morelos. Repositorio de la Univerisidad Aut�noma Del Estado de Morelos, MORELOS. Obtenido de http://riaa.uaem.mx/handle/20.500.12055/3256

  11.            Torres Mateu, N. (2023). T�cnicas para recuperaci�n de aguas contaminadas por hidrocarburos. [Universidad de Am�rica ]. Repositorio Institucional. Obtenido de https://hdl.handle.net/20.500.11839/9207

  12.            Ure�o Garc�a, U. (2023). Evaluaci�n de la estimulaci�n de lipasa de Ricinus Communis L. con surfactante y lombricomposta comercial en la remediaci�n de un suelo contaminado con aceite residual automotriz. [Tesis de Ingenier�a, Instituto Polit�cnico Nacional]. Repositorio Institucional. Obtenido de http://tesis.ipn.mx/handle/123456789/31828

  13.            Romero, E., Cobo, J., P�rez, K., & Polo, L. (2020). PREPARACI�N DE MEDIOS DE CULTIVO Y MANEJO DEL AUTOCLAVE[informe, Universidad de sucre]. Quito. Obtenido de https://d1wqtxts1xzle7.cloudfront.net/60248448/Informe_4_MICROBIOLOGIA_-_PREPARACION_DE_MEDIOS_DE_CULTIVO_Y_MANEJO_DEL_AUTOCLAVE20190809-38390-3e00tt-libre.pdf?1565385193=&response-content-disposition=inline%3B+filename%3DInforme_4_MICROBIOLOGIA_PREPARACI

  14.            Guerra Yepes, L. M. (2019). Avances en la remediaci�n biol�gica del mercurio: hongos macrosc�picos como potenciales agentes de biorremediaci�n.[Trabajo de grado - Maestr�a,Univerisidad Nacional de Colombia]. Repositorio instucional. Obtenido de https://repositorio.unal.edu.co/handle/unal/77625

  15.            C�rdenas Rodr�guez, A. (2021). Planificaci�n de las acciones b�sicas de respuestas en caso de incidentes, accidentes o estados de emergencias ambientales de la Universidad de la Costa. [Tesis de Pregrado, Universidad de la Costa]. Repositorio Institucional. Obtenido de https://hdl.handle.net/11323/8873

  16.            Mendoza Lombana, S. (2022). Validaci�n de estrategias profil�cticas en larvicultura del camar�n Penaeus vannamei en la provincia de Santa Elena, Mar Bravo. [Universidad Estatal Pen�nsula de Santa Elena]. Repositorio Institucional. Obtenido de https://repositorio.upse.edu.ec/handle/46000/8074

  17.            Rodr�guez, P., & Arenas Roberto. (2018). Hans Christian Gram y su tinci�n. Dermatolog�a Cosm�tica, M�dica y Quir�rgica., 16(2), 2. doi:https://www.medigraphic.com/cgi-bin/new/resumen.cgi?IDARTICULO..

  18.            S�nchez S�nchez, F. (2020). Evaluaci�n de las estrategias utilizadas por las industrias petroleras (Ecopetrol-Pacific Rubiales Energy) en la resoluci�n de conflictos socioambientales. Estudio de caso del municipio de Granada � (21) Meta periodo 2014-2018. [Tesis de Maestr�a]. Universidad de Ciencias Aplicadas y Ambientales. Obtenido de https://repository.udca.edu.co/handle/11158/3600

  19.            S�nchez Riofrio, C. D. (2022). Identificaci�n de microorganismos mediante la captura en dos terrazas agr�colas en el Campus Salache, Latacunga, Cotopaxi.[Tesis de pregrado, Universidad t�cnica de Cotopaxi]. Identificaci�n de microorganismos mediante la captura en dos terrazas agr�colas en el Campus Salache, Latacunga, Cotopaxi. Repositorio Digital Universidad T�cnica de Cotopaxi , Latacunga. Obtenido de http://repositorio.utc.edu.ec/handle/27000/9604

  20.            Science Photo Library. (29 de abril de 2013). Alamy Foto de stock (fotograf�a). Obtenido de Alamy: www.alamy.es

  21.            S�nchez Salao, M. (2021). Dise�o de un filtro de aire para reducir di�xido de carbono emitido por veh�culos automotores de gasolina. [Tesis de Ingenier�a,Escuela Superior Polit�cnica de Chimborazo ]. Repositorio Institucional. Obtenido de http://dspace.espoch.edu.ec/handle/123456789/15320

  22.            Manrique, M., & Guisella, E. (2021). Tecnolog�as amigables con el ambiente para la recuperaci�n de suelo urbano contaminado por hidrocarburos en un taller mec�nico, Chilca[Tesis de pregrado, Universidad Nacional del Centro del Per�]. Tecnolog�as amigables con el ambiente para la recuperaci�n de suelo urbano contaminado por hidrocarburos en un taller mec�nico, Chilca. Repositorio de la Universidad Nacional del Centro del Per�, Chilca. Obtenido de http://hdl.handle.net/20.500.12894/7229

  23.            Sossa L�pez, L. (2019). Dise�o de protocolo de manejo seguro de sustancias qu�micas para el Centro de Dise�o e Innovaci�n Tecnol�gica Industrial, Sena Dosquebradas. [Universidad Libre]. Repositorio Institucional. Obtenido de https://hdl.handle.net/10901/20062

  24.            Vergaray Ruiz, V. (2022). Aplicaci�n de la t�cnica del microcosmo para evaluar la capacidad de los lodos residuales como biorremediadores de suelos contaminados con hidrocarburos[Tesis de licenciatura, Universidad Privada Del Norte ]. Aplicaci�n de la t�cnica del microcosmo para evaluar la capacidad de los lodos residuales como biorremediadores de suelos contaminados con hidrocarburos. Repositorio institucional UPN, Lima. Obtenido de https://hdl.handle.net/11537/31244

  25.            Villa Morales, L. (2019). Aplicaci�n de sistemas de biorremediaci�n en residuos acuosos generados por estaciones de servicio: Caso Planta Senkata Y.P.F.B. [Proyecto de Grado, Universidad Mayor de San Andr�s]. Repositorio Institucional. Obtenido de http://repositorio.umsa.bo/xmlui/handle/123456789/32257

  26.            Zimmermann, R. (2022). Quemaduras qu�micas y carbonizaci�n: efectos sobre tatuajes y su incidencia en la identificaci�n de cad�veres. [Universidad Fasta]. Repositorio Institucional. Obtenido de http://redi.ufasta.edu.ar:8082/jspui/handle/123456789/683

  27.            Tamba Pineda, M. A. (2023). AN�LISIS DE LA CASCARILLA DE ARROZ (Oryza sativa) COMO BIOADSORBENTE DE LA CONCENTRACI�N DE CONTAMINANTES DEL EFLUENTE DE UNA LUBRICADORA. [Tesis de Ingenier�a, Universdiad Agraria del Ecuador]. Repositorio Institucional, Ecuador. Obtenido de https://cia.uagraria.edu.ec/Archivos/TAMBA%20PINEDA%20MARCELO%20AARON.pdf

  28.            Tipantu�a Chiluisa, N. (2020). Cin�tica del crecimiento de microorganismos durante el proceso de fermentaci�n de una bebida ancestral elaborada a partir de chonta (Bactris gasipaes ). [Tesis de ingenier�a, Universidad T�cnica de Cotopaxi ]. Repositorio Institucional. Obtenido de http://repositorio.utc.edu.ec/handle/27000/6705

 

 

 

 

 

 

 

 

 

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