Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro
Digital
- Autores:
-
Osorio-Acero, Juan Nicolás
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2023
- Institución:
- Universidad de Santander
- Repositorio:
- Repositorio Universidad de Santander
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.udes.edu.co:001/9278
- Palabra clave:
- Biorremediación
Asfaltenos
Mineralización
qPCR
Abundancia Relativa
Bioremediation
Asphaltenes
Mineralization
Relative Abundance
- Rights
- openAccess
- License
- http://purl.org/coar/access_right/c_abf2
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| dc.title.spa.fl_str_mv |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| dc.title.translated.none.fl_str_mv |
Evaluation of Changes in the Abundance of the Gene Coding for the Enzyme Catechol-2,3-dioxygenase (catA) During the Asphaltene Biodegradation Process in an in vitro System |
| title |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| spellingShingle |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro Biorremediación Asfaltenos Mineralización qPCR Abundancia Relativa Bioremediation Asphaltenes Mineralization Relative Abundance |
| title_short |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| title_full |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| title_fullStr |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| title_full_unstemmed |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| title_sort |
Evaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitro |
| dc.creator.fl_str_mv |
Osorio-Acero, Juan Nicolás |
| dc.contributor.advisor.none.fl_str_mv |
Valdivieso-Quintero, Wilfredo |
| dc.contributor.author.none.fl_str_mv |
Osorio-Acero, Juan Nicolás |
| dc.contributor.jury.none.fl_str_mv |
Osorio-Márquez, Jorge Daniel Agualimpia-Valderrama, Bayron Enrique |
| dc.subject.proposal.spa.fl_str_mv |
Biorremediación Asfaltenos Mineralización qPCR Abundancia Relativa |
| topic |
Biorremediación Asfaltenos Mineralización qPCR Abundancia Relativa Bioremediation Asphaltenes Mineralization Relative Abundance |
| dc.subject.proposal.eng.fl_str_mv |
Bioremediation Asphaltenes Mineralization Relative Abundance |
| description |
Digital |
| publishDate |
2023 |
| dc.date.accessioned.none.fl_str_mv |
2023-09-25T14:02:56Z |
| dc.date.available.none.fl_str_mv |
2023-09-25T14:02:56Z |
| dc.date.issued.none.fl_str_mv |
2023-07-27 |
| dc.type.none.fl_str_mv |
Trabajo de grado - Pregrado |
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http://purl.org/coar/resource_type/c_7a1f |
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Text |
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info:eu-repo/semantics/bachelorThesis |
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http://purl.org/redcol/resource_type/TP |
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Bucaramanga, Colombia |
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T 33.23 O867e |
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Repositorio Digital Universidad de Santander |
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https://repositorio.udes.edu.co |
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https://repositorio.udes.edu.co/handle/001/9278 |
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Bucaramanga, Colombia T 33.23 O867e Repositorio Digital Universidad de Santander |
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https://repositorio.udes.edu.co https://repositorio.udes.edu.co/handle/001/9278 |
| dc.language.iso.none.fl_str_mv |
spa |
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Adebayo, O. y Chiomam, A. (2019). Petroleum Sludge Treatment and Disposal: A Review. Environmental Engineering Research, 24(2), 191-201 Adebiyi, F. (2021). An Insight Into Asphaltene Precipitation, Deposition and Management Stratagems in Petroleum Industry. Journal of Pipeline Science and Engineering, 1(4), 419-427 Afanasjeva, N. (2014). Conversión Pirolítica de los Concentrados Resino-Asfalténicos de los Crudos Pesados. Revista ION, 27(2), 71-86 Ali, N., Khanafer, M., Al-Awadhi, H., Radwan, S. (2020). Self-cleaning of Very Heavily Oil-Polluted Sites Proceeds Even Under Heavy-Metal Stress While Involved Bacteria Exhibit Bizarre Pleomorphism. Ecotoxicology and Environmental Safety, 200 Alshareef, A. H. (2020). Asphaltenes: Definition, Properties, and Reactions of Model Compounds. Energy Fuels, 34 (1), 3–16 Azubuike, C. C., Chikere, C. B., & Okpokwasili, G. C. (2016). Bioremediation Techniques–Classification Based on Site of Application: Principles, Advantages, Limitations and Prospects. World Journal of Microbiology and Biotechnology, 32(11) Bochner de Araujo, S., Merola, M., Vlassopoulos, D., Gerald G. (2017). Droplet Coalescence and Spontaneous Emulsification in the Presence of Asphaltene Adsorption. Fuller Langmuir, 33 (40), 10501-10510 Botes, M., de Kwaadsteniet, M. & Cloete, T.E. (2013). Application of Quantitative PCR for the Detection of Microorganisms in Water. Anal Bioanal Chem, 405, 91–108 Boussingault, J. B. (1837). Mémoire sur la Composition des Bitumes. Ann Chim Phys, 64, 141–151 Bressler, D. y Gray, M. (2003). Transport and Reaction Processes in Bioremediation of Organic Contaminants. 1. Review of Bacterial Degradation and Transport. International Journal of Chemical Reactor Engineering, 1(1) Degnarain, N. (2020). How Satellites Tracked the Fateful Journey of the Ship That led to Mauritius’ Worst Oil Spill Disaster. Forbes. https://www.forbes.com/sites/nishandegnarain/2020/08/09/how-satellites-traced-the-fateful-journey-of-the-ship-that-led-to--mauritius-worst-oil-spill-disaster/?sh=235ac8705b42 Desai, C., Jain, K., Patel, B. Madamwar, D. (2009). Efficacy of Bacterial Consortium-AIE2 for Contemporaneous Cr(VI) and azo dye Bioremediation in Batch and Continuous Bioreactor Systems, Monitoring Steady-State Bacterial Dynamics Using qPCR Assays. Biodegradation 20, 813–826 Dymond, J. S. (2013). Explanatory Chapter: Quantitative PCR. En J. Lorsch (Ed.), Methods in Enzymology (Vol. 529, pp. 279-289). Academic Press Fakher, S., Ahdaya, M., Elturki, M. (2020). Critical Review of Asphaltene Properties and Factors Impacting its Stability in Crude oil. J Petrol Explor Prod Technol 10, 1183–1200 Fink, J. (2021). Enhanced oil Recovery. Petroleum Engineer's Guide to Oil Field Chemicals and Fluids (Third Edition). (643-731). Gulf Professional Publishing Freund, H., Walters, C. C., Kelemen, S. R., Siskin, M., Gorbaty, M. L.; Curry, D. J., Bence, A. E. (2007). Predicting oil and gas Compositional Yields via Chemical Structure-Chemical Yield Modeling (CS-CYM): Part 1 - Concepts and Implementation. Org. Geochem, 38 (2), 288– 305 Fuller, M. E. (2005). POLLUTANTS | Effects on Microorganisms. Encyclopedia of Soils in the Environment, 258–264 Galitskaya, P., Biktasheva, L., Blagodatsky, S. (2021). Response of Bacterial and Fungal Communities to High Petroleum Pollution in Different Soils. Sci Rep, 11, 164 Garzón, J. M. , Rodríguez-Miranda, J. P. y Hernández-Gómez, C. (2017). Revisión del Aporte de la Biorremediación Para Solucionar Problemas de Contaminación y su Relación con el Desarrollo Sostenible. Rev. Univ. Salud, 19(2), 309-318 Goual, L. (2012). Petroleum Asphaltenes, Crude oil Emulsions–Composition Stability and Characterization. ISBN, 978-953 Guy RA, Payment P, Krull UJ, Horgen PA (2003). Real-Time PCR for Quantification of Giardia and Cryptosporidium in Environmental Water Samples and Sewage. Appl Environ Microbiol, 69(9), 5178–5185 Hidalgo, K. J., Sierra-García, I. N., Dellagnezzem, B. M., De Oliviera, V. M. (2020). Metagenomic Insights into the Mechanisms for Biodegradation of Polycyclic Aromatic Hidrocarbons in the oil Supply Chain. Frontiers in Microbiology, 11 Hernández-López, E. L., Ayala, M., & Vazquez-Duhalt, R. (2015). Microbial and Enzymatic Biotransformations of Asphaltenes. Petroleum Science and Technology, 33(9), 1017–1029 Hernández-López, E. Lorena, Perezgasga, L., Huerta-Saquero, A., Mouriño-Pérez, R., & VazquezDuhalt, R. (2016). Biotransformation of Petroleum Asphaltenes and High Molecular Weight Polycyclic Aromatic Hydrocarbons by Neosartorya fischeri. Environmental Science and Pollution Research, 23(11), 10773–10784 Iwamoto, T., Tani, K., Nakamura, K., Suzuki, Y., Kitagawa, M., Eguchi, M., Nasu, M. (2000). Monitoring Impact of in situ Biostimulation Treatment on Groundwater Bacterial Community by DGGE. FEMS Microbiology Ecology, 32, 129–141 Kamkar, M. y Giovanniantonio, N. (2021) A Review on Novel Applications of Asphaltenes: A Valuable Waste. Fuel, 285, 119272 Kjelleberg, S. y Hermansson, M. (1984). Starvation-Induced Effects on Bacterial Surface Characteristics. Applied and environmental microbiology, 48 (3), 497-503 Leahy, J. G., & Colwell, R. R. (1990). Microbial Degradation of Gydrocarbons in the Environment. Microbiological reviews, 54(3), 305–315 Le Goff, O., Godon, J. P, Steyer, J. P., Wéry, N. (2011). New Specific Indicators for qPCR Monitoring of Airborne Microorganisms Emitted by Composting Plants. Atmospheric Environment, 45, 5342-5350 Li, P., Sun, T., Stagnitti, F., Zhang, C., Zhang, H., Xiong, X., Allinson, M. (2002). Field-Scale Bioremediation of Soil Contaminated with Crude Oil. Environmental Engineering Science, 19(5), 277–289 Plata-Gryl, M., Momotko, M., Makowiec, S., Boczkaj, G. (2019). Highly Effective Asphaltene Derived Adsorbents for Gas Phase Eemoval of Volatile Organic Compounds. Separation and Purification Technology, 224, 315-321 Maddocks, S. y Jenkins, R. (2017). Quantitative PCR: Things to Consider. Understanding PCR (pp. 45-52). Academic Press. Margesin R, Zimmerbauer A, Schinner F. (2000). Monitoring of Bioremediation by Soil Biological Activities. Chemosphere, 40(4), 339-46 Martínez-Sepúlveda, J.A. y Reinaldo-Casallas, M. 2018. Contaminación y Remediación de Suelos en Colombia. Aplicación a la Minería de Oro. 1ra edición. Ediciones EAN. Universidad EAN. Bogotá D.C. http://editorial.universidadean.edu.co/acceso-abierto/contaminacion-y-remediacion-de-suelos-en-colombia.pdf Mendizabala A, Holmes J, Ortiz N. (2021). A Hotspot Analysis of Critical Hydrocarbons Infrastructure in Colombia: ELN (Ejército de Liberación Nacional) and FARC (Fuerzas Armadas Revolucionarias de Colombia) Attacks on Colombian Pipelines Mojiri, A., Zhou, J. L., Ohashi, A., Ozaki, N., Kindaichi, T. (2019). Comprehensive Review of Polycyclic Aromatic Hydrocarbons in Water Sources, their Effects and Treatments. Science of The Total Environment, 696, 133971 Moriarty, D.J.W. (1986). Measurement of Bacterial Growth Rates in Aquatic Systems from Rates of Nucleic Acid Synthesis. In: Marshall, K.C. (eds) Advances in Microbial Ecology. Advances in Microbial Ecology, vol 9. Springer Muthukamalam, S., Sivagangavathi, S., Dhrishya, D., Sudha Rani, S. (2017). Characterization of Dioxygenases and Biosurfactants Produced by Crude Oil Degrading Soil Bacteria. Brazilian Journal of Microbiology, 48(4), 637–647 Naranjo-Briceño, L., Pernía, B., Perdomo, T., González, M., Inojosa, Y., De Sisto, Á., Urbina, H., León, V. (2019). Potential Role of Extremophilic Hydrocarbonoclastic Fungi for Extra-Heavy Crude Oil Bioconversion and the Sustainable Development of the Petroleum Industry. En Tiquia-Arashiro, S., Grube, M. (eds) Fungi in Extreme Environments: Ecological Role and Biotechnological Significance (559-586). Springer Nelson-Smith, A. (1979). Effect of Oil Spills on Land and Water. The prevention of oil pollution. Graham and Trotman Limited Nzila, A. y Musa, M. M. (2021) Current Knowledge and Future Challenges on Bacterial Degradation of the Highly Complex Petroleum Products Asphaltenes and Resins. Front. Environ. Sci., 9 Pernía, B., Demey, J. R., Inojosa, Y., Naranjo, L. (2012). Biodiversidad y Potencial Hidrocarbonoclástico de Hongos Aislados de Crudo y sus Derivados: Un Meta-Análisis. Revista Latinoamericana de Biotecnología Ambiental Algal, 3(1), 1-39 Posow. (2016). Oil Spill Waste Management Manual. https://www.posow.org/documentation/wasteweb.pdf Pritchard, P.H., Mueller, J.G., Rogers, J.C., Kremer, F.V., Glaser, J.A. (1992). Oil Spill Bioremediation: Experiences, Lessons and Results from the Exxon Valdez Oil Spill in Alaska. Biodegradation, 3 Qu, W., Guo, Y., Shen, W., Li, W. (2016). Using Asphaltene Supermolecules Derived from Coal for the Preparation of Efficient Carbon Electrodes for Supercapacitors. The Journal of Physical Chemistry, 120 (28), 15105-15113 Rodrigues, E., Kalks, K., Tótola. M. (2015). Prospect, Isolation, and Characterization of Microorganisms for Potential Use in Cases of Oil Bioremediation along the Coast of Trindade Island, Brazil. Journal of Environmental Management, 156, 15-22 Ruiz, O. N., Brown, L. M., Radwan, O., Bowen, L., Gunasekera, T., Mueller, S., West, Z., Striebich, R. (2021). Metagenomic Characterization Reveals Complex Association of Soil Hydrocarbon-Degrading Bacteria. International Biodeterioration & Biodegradation, 157 Sakshi, Haritash, A.K. (2020). A Comprehensive Review of Metabolic and Genomic Aspects of PAH-Degradation. Arch Microbiol 202, 2033–2058 Sales da Silva, I., Gomes de Almeida, F. C., Padilha da Rocha, N., Casazza A., (2020). Soil Bioremediation: Overview of Technologies and Trends. Energies, 13, 1–25 Sangkharak, K., Choonut, A., Rakkan, T. et al. (2020). The Degradation of Phenanthrene, Pyrene, and Fluoranthene and Its Conversion into Medium-Chain-Length Polyhydroxyalkanoate by Novel Polycyclic Aromatic Hydrocarbon-Degrading Bacteria. Curr Microbiol 77, 897–909 Sei, K., Asano, K.-I., Tateishi, N., Mori, K., Ike, M., Fujita, M. (1999). Design of PCR Primers and Gene Probes for the General Detection of Bacterial Populations Capable of Degrading Aromatic Compounds Via Catechol Cleavage Pathways. Journal of Bioscience and Bioengineering, 88(5), 542–550 Semple, K., Morriss, A., Paton, G. (2003). Bioavailability of Hydrophobic Organic Contaminants in Soils: Fundamental Concepts and Techniques for Analysis. European Journal of Soil Science, 54, 809 – 818 Siddiqui, M. N. (2017). Preparation and Properties of Polypropylene-Asphaltene Composites. Polym Compos, 38(9), 1957–63 Shafeeq, M., Kokub, D., Khalid, Z.M. (1989). Degradation of Different Hydrocarbons and Production of Biosurfactant by Pseudomonas aeruginosa Isolated from Coastal Waters. World J Microbiol Biotechnol, 5, 505–510 Sharma, Neha. (2019). Microbes and their Secondary Metabolites: Agents in Bioremediation of Hydrocarbon Contaminated Site. Archives of Petroleum & Environmental Biotechnology,4 Sun, K., Song, Y., He, F., Jing, M., Tang, J., Liu, R. (2021). A review of Human and Animals Exposure to Polycyclic Aromatic Hydrocarbons: Health Risk and Adverse Effects, Photo-Induced Toxicity and Regulating Effect of Microplastics. Science of The Total Environment, 773, 145403 Tavassoli, T., Mousavi, S.M., Shojaosadati, S.A., Salehizadeh, H. (2012). Asphaltene Biodegradation Using Microorganisms Isolated from Oil Samples. Fuel, 93, 142-148 Ultee, E., Ramijan, K., Dame, R. T., Briegel, A., & Claessen, D. (2019). Stress-Induced Adaptive Morphogenesis in Bacteria. Advances in Microbial Physiology, 97–141 Varjani, S. J., & Gnansounou, E. (2017). Microbial Dynamics in Petroleum Oilfields and their Relationship with Physiological Properties of Petroleum Oil Reservoirs. Bioresour. Technol. 245, 1258–1265 Wang, X., Cai, T., Wen, W., Zhang, Z. (2018). Effect of Biosurfactant on Biodegradation of Heteroatom Compounds in Heavy Oil. Fuel, 230, 418-429 Waychal, Y., Gawas, S. and Barage, S. (2022). Bioremediation of Petroleum-Contaminated Soil. Advances in Bioremediation and Phytoremediation for Sustainable Soil Management: Principles, Monitoring and Remediation. Springer Yap, H. S., Zakaria, N. N., Zulkharnain, A., Sabri, S., Gomez-Fuentes, C., & Ahmad, S. A. (2021). Bibliometric Analysis of Hydrocarbon Bioremediation in Cold Regions and a Review on Enhanced Soil Bioremediation. Biology, 10(5), 354 Zachos, E. (2018). Oil Spill Kills More Than 2,400 Animals. National Geographic. https://www.nationalgeographic.com/animals/article/oil-spill-colombia-animals-killed-spd Zafra, G. A. (2014). Microcosm Evaluation and Metagenomic Analysis of the Bioremediation of Soils Contaminated with PAHS by Microbial Consortia. [Tesis doctoral, Instituto Politécnico Nacional]. Repositorio institucional del Instituto Politécnico Nacional http://rdcb.cbg.ipn.mx/handle/20.500.12273/736 Zafra, G., Taylor, T. D., Absalón, A. E., & Cortés-Espinosa, D. V. (2016). Comparative Metagenomic Analysis of PAH Degradation in Soil by a Mixed Microbial Consortium. Journal of Hazardous Materials, 318, 702–710 Zargar, A. N., Kumar, A., Sinha, A., Kumar, M., Skiadas, I., Mishra, S., Srivastava, P. (2021). Asphaltene Biotransformation for Heavy Oil Upgradation. AMB Express, 11(1), 127 |
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Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0) |
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Universidad de Santander |
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Bucaramanga |
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Facultad de Ciencias Agrícolas y Veterinarias |
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Bucaramanga, Colombia |
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Microbiología Industrial |
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Universidad de Santander |
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Universidad de Santander |
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Valdivieso-Quintero, Wilfredo8a364218-619a-4695-a854-44b943784e23-1Osorio-Acero, Juan Nicolás83b9b159-a09f-425b-b461-6dae33d832d0-1Osorio-Márquez, Jorge Daniel479806cb-7908-4c07-aebb-e75bd4014037600Agualimpia-Valderrama, Bayron Enrique88ec3c05-4636-40b3-b2ad-e2f2f07af405-12023-09-25T14:02:56Z2023-09-25T14:02:56Z2023-07-27DigitalUna de las actividades de hoy en día que se encuentra en constante crecimiento es la exploración y explotación de hidrocarburos. El petróleo es la principal fuente energética y de comercio en la mayoría de los países. Según los informes de Ecopetrol, en Colombia se producen alrededor de 866.488 barriles diarios, de los que cerca del 42% son de crudo pesado. El crudo pesado, en su mayoría compuesto por asfaltenos, presentan daños perjudiciales al medio ambiente y a la biodiversidad en los ecosistemas. El objetivo de este estudio fue estudiar los cambios en la abundancia del gen catA de un consorcio microbiano conformado por Pseudomonas stutzeri, Rhodococcus corynebacterioides y Rhodococcus qingshengii, luego de la exposición a asfaltenos y petróleo crudo ligero. Para ello, se determinó los niveles de producción de CO2 por parte del consorcio bacteriano durante la biodegradación de asfaltenos y petróleo crudo ligero, con el fin de monitorear el crecimiento y la actividad metabólica del consorcio. Para esto, se realizó una extracción de ADNg total, al cual se realizó qPCR con el propósito de cuantificar los genes ARNr 16S y catA del consorcio microbiano en presencia de asfaltenos y petróleo crudo ligero. Por último, utilizando el método de Livak (2−) se estimó la abundancia relativa del gen catA durante la biodegradación de asfaltenos por parte del consorcio bacteriano. Los resultados sugieren que la actividad metabólica del consorcio y los cambios del gen catA son independientes a la cantidad de copias del gen ARNr 16S. En conclusión, se evidenció una mayor abundancia relativa del gen catA en el tratamiento con asfaltenos en comparación con el tratamiento con petróleo crudo ligero.One of today's ever-growing activities is the exploration and exploitation of hydrocarbons. Oil is the main source of energy and trade in most countries. According to Ecopetrol reports, Colombia produces about 866,488 barrels per day, of which about 42% is heavy crude oil. Heavy crude oil, mostly composed of asphaltenes, is harmful to the environment and biodiversity in ecosystems. The objective of this study was to study the changes in the abundance of the catA gene of a microbial consortium composed of Pseudomonas stutzeri, Rhodococcus corynebacterioides and Rhodococcus qingshengii, after exposure to asphaltenes and light crude oil. For this purpose, the levels of CO2 production by the bacterial consortium during the biodegradation of asphaltenes and light crude oil were determined to monitor the growth and metabolic activity of the consortium. For this, a total gDNA extraction was performed and qPCR was performed with the purpose of quantifying the 16S rRNA and catA genes of the microbial consortium in the presence of asphaltenes and light crude oil. Finally, using the method of Livak (2^(-Ct)) the relative abundance of the catA gene during asphaltene biodegradation by the bacterial consortium was estimated. The results suggest that the metabolic activity of the consortium and catA gene changes are independent of 16S rRNA gene copy number. In conclusion, a higher relative abundance of the catA gene was evident in the asphaltene treatment compared to the light crude oil treatment.PregradoMicrobiólogo IndustrialIntroducción.......17 Planteamiento Problema.......20 Justificación.......23 Objetivos.......25 Objetivo General.......25 Objetivos Específicos.......25 Marco Teórico.......26 Asfaltenos.......26 Composición de los Asfaltenos.......26 Contaminación por Petróleo Crudo y Asfaltenos.......27 Casos de Contaminación por Petróleo Crudo y Asfaltenos.......28 Efectos de los Asfaltenos al Medio Ambiente.......28 Disposición Final de los Asfaltenos.......29 Biorremediación por Bacterias.......30 Ensayos de Mineralización.......31 La Técnica de Reacción en Cadena de la Polimerasa Cuantitativa (qPCR).......31 Seguimiento de Poblaciones por qPCR.......32 Metodología.......34 Consorcio Microbiano.......34 Extracción de ADN Genómico (ADNg).......34 Revisión Literaria de Oligonucleótidos Para los Genes ARNr 16S y catA.......35 Reacción en Cadena de la Polimerasa (PCR del Inglés “Polimerase Chain Reaction”).......36 Reacción en Cadena de la Polimerasa en Tiempo Real (qPCR del Inglés “Quantitative Polymerase Chain Reaction”).......37 Ensayo de Biodegradación de Asfaltenos in vitro.......37 Resultados.......40 Caracterización Morfológica de los Microorganismos.......40 Extracción de ADNg.......42 Amplificación por Reacción en Cadena de Polimerasa.......42 Ensayo de Biodegradación de Asfaltenos en Microcosmos.......44 Cuantificación ADNg.......44 Producción de CO2 en Microcosmos.......45 Amplificación por PCR en Tiempo Real.......47 Análisis Abundancia Relativa del Gen catA.......50 Discusión de Resultados.......52 Conclusiones.......59 Recomendaciones.......60 Referencias Bibliográficas.......6169 papplication/pdfBucaramanga, ColombiaT 33.23 O867eRepositorio Digital Universidad de Santanderhttps://repositorio.udes.edu.cohttps://repositorio.udes.edu.co/handle/001/9278spaUniversidad de SantanderBucaramangaFacultad de Ciencias Agrícolas y VeterinariasBucaramanga, ColombiaMicrobiología IndustrialAdebayo, O. y Chiomam, A. 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Asphaltene Biotransformation for Heavy Oil Upgradation. AMB Express, 11(1), 127Derechos Reservados - Universidad de Santander, 2023. Al consultar y hacer uso de este recurso, está aceptando las condiciones de uso establecidas por los autores.info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Atribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)https://creativecommons.org/licenses/by-nc-nd/4.0/BiorremediaciónAsfaltenosMineralizaciónqPCRAbundancia RelativaBioremediationAsphaltenesMineralizationRelative AbundanceEvaluación de Cambios en la Abundancia del gen Codificante para la Enzima Catecol-2,3-dioxigenasa (catA) Durante el Proceso de Biodegradación de Asfaltenos en un Sistema in vitroEvaluation of Changes in the Abundance of the Gene Coding for the Enzyme Catechol-2,3-dioxygenase (catA) During the Asphaltene Biodegradation Process in an in vitro SystemTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_71e4c1898caa6e32Textinfo:eu-repo/semantics/bachelorThesishttp://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/submittedVersionTodas las AudienciasPublicationLICENSElicense.txtlicense.txttext/plain; charset=utf-815543https://repositorio.udes.edu.co/bitstreams/e6edce83-324a-4574-b672-2e2cc4b6a3db/download73a5432e0b76442b22b026844140d683MD55ORIGINALLabel.pngLabel.pngimage/png113476https://repositorio.udes.edu.co/bitstreams/2f409401-96ef-440d-beb1-c73c8fca644e/downloade5cc5335cf00666cc1aba622f9d9336dMD511Evaluación_de_Cambios_en_la_Abundancia_del_Gen_Codificante para_la_Enzima_Catecol-2,3-dioxigenasa_ (catA)_Durante_el_Proceso_de_Biodegradación_de_Asfaltenos_en_un_Sistema_in_vitro.pdfEvaluación_de_Cambios_en_la_Abundancia_del_Gen_Codificante para_la_Enzima_Catecol-2,3-dioxigenasa_ (catA)_Durante_el_Proceso_de_Biodegradación_de_Asfaltenos_en_un_Sistema_in_vitro.pdfapplication/pdf1386685https://repositorio.udes.edu.co/bitstreams/dd13a3fe-6d10-4623-8ae6-a8cbe0e548ed/downloadb3832b88e775ebc16c655b8b3b79463cMD510Similitud de texto.pdfSimilitud de texto.pdfapplication/pdf1403045https://repositorio.udes.edu.co/bitstreams/3e9f4775-e16b-44d5-bf83-35ea8c907173/download81ecbd63678e2af7ec80c30941d078b0MD57THUMBNAILLabel.png.jpgLabel.png.jpgGenerated Thumbnailimage/jpeg12524https://repositorio.udes.edu.co/bitstreams/1264752d-f67a-4d03-99d1-4928fde310a8/download1cd016a82701bfb5873e31f5a0362979MD512Evaluación_de_Cambios_en_la_Abundancia_del_Gen_Codificante para_la_Enzima_Catecol-2,3-dioxigenasa_ (catA)_Durante_el_Proceso_de_Biodegradación_de_Asfaltenos_en_un_Sistema_in_vitro.pdf.jpgEvaluación_de_Cambios_en_la_Abundancia_del_Gen_Codificante para_la_Enzima_Catecol-2,3-dioxigenasa_ (catA)_Durante_el_Proceso_de_Biodegradación_de_Asfaltenos_en_un_Sistema_in_vitro.pdf.jpgGenerated Thumbnailimage/jpeg7887https://repositorio.udes.edu.co/bitstreams/de3ab213-5836-44ba-a564-b4e4978088cf/downloadf3655226468796dbd9e898abb6fbdd4bMD514Similitud de texto.pdf.jpgSimilitud de texto.pdf.jpgGenerated Thumbnailimage/jpeg9157https://repositorio.udes.edu.co/bitstreams/77fc2b2f-d431-49dc-bcf9-ddaba4b26be4/download57dcfe32d90373288fcb4bad6d78fcbbMD516TEXTEvaluación_de_Cambios_en_la_Abundancia_del_Gen_Codificante para_la_Enzima_Catecol-2,3-dioxigenasa_ (catA)_Durante_el_Proceso_de_Biodegradación_de_Asfaltenos_en_un_Sistema_in_vitro.pdf.txtEvaluación_de_Cambios_en_la_Abundancia_del_Gen_Codificante para_la_Enzima_Catecol-2,3-dioxigenasa_ (catA)_Durante_el_Proceso_de_Biodegradación_de_Asfaltenos_en_un_Sistema_in_vitro.pdf.txtExtracted texttext/plain95482https://repositorio.udes.edu.co/bitstreams/f4ebae2c-71aa-43d6-a952-e2f062ea9d61/download46da8871a42f31d64fa93dce6bdc0ae9MD513Similitud de texto.pdf.txtSimilitud de texto.pdf.txtExtracted texttext/plain100762https://repositorio.udes.edu.co/bitstreams/f2dd348e-fcde-4877-8eee-bf7338a1cce7/downloadaf2d7df8f2c56f511d7f5444badc8ca4MD515001/9278oai:repositorio.udes.edu.co:001/92782023-09-26 03:03:21.787https://creativecommons.org/licenses/by-nc-nd/4.0/Derechos Reservados - Universidad de Santander, 2023. 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