Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio
H. pluvialis es una microalga utilizada para la producción de astaxantina, este es un betacaroteno y antioxidante en la industria. El objetivo del trabajo es utilizar diferentes factores de estrés para obtener una mayor producción de astaxantina, la microalga se cultivó en medio RM bajo condiciones...
- Autores:
-
Rodríguez Romero, Laura Johanna
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2019
- Institución:
- Colegio Mayor de Cundinamarca
- Repositorio:
- Repositorio Colegio Mayor de Cundinamarca
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unicolmayor.edu.co:unicolmayor/3709
- Acceso en línea:
- https://repositorio.unicolmayor.edu.co/handle/unicolmayor/3709
- Palabra clave:
- factores de estrés
estrés salino
luz
microalga
carotenoides
- Rights
- closedAccess
- License
- Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2019
| id |
UCOLMAYOR2_097051f7cac2ad2c476d404307918c8d |
|---|---|
| oai_identifier_str |
oai:repositorio.unicolmayor.edu.co:unicolmayor/3709 |
| network_acronym_str |
UCOLMAYOR2 |
| network_name_str |
Repositorio Colegio Mayor de Cundinamarca |
| repository_id_str |
|
| dc.title.spa.fl_str_mv |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| title |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| spellingShingle |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio factores de estrés estrés salino luz microalga carotenoides |
| title_short |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| title_full |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| title_fullStr |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| title_full_unstemmed |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| title_sort |
Producción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodio |
| dc.creator.fl_str_mv |
Rodríguez Romero, Laura Johanna |
| dc.contributor.advisor.none.fl_str_mv |
Camacho Kurmen, Judith Elena |
| dc.contributor.author.none.fl_str_mv |
Rodríguez Romero, Laura Johanna |
| dc.subject.lemb.none.fl_str_mv |
factores de estrés estrés salino luz |
| topic |
factores de estrés estrés salino luz microalga carotenoides |
| dc.subject.proposal.spa.fl_str_mv |
microalga carotenoides |
| description |
H. pluvialis es una microalga utilizada para la producción de astaxantina, este es un betacaroteno y antioxidante en la industria. El objetivo del trabajo es utilizar diferentes factores de estrés para obtener una mayor producción de astaxantina, la microalga se cultivó en medio RM bajo condiciones de pH 6,8, temperatura 20±2oC, aire filtrado, iluminación con lámparas blancas con 20h luz/4h oscuridad, de 70 μE m−2 s −1 con diferentes concentraciones de acetato de sodio y cloruro de sodio adicionadas a diferentes tiempos. Este estudio se realizó durante 56 días. Se determinó el crecimiento celular, el análisis de los cambios morfológicos y la cuantificación de astaxantina y clorofila por el método de espectrofotometría. Se realizó un análisis estadístico utilizando ANOVA (95%). Se evidencio una mayor producción de astaxantina al ensayo adicionado con 1,6 g/L de acetato de sodio y 6,4 g/L de cloruro de sodio. Un 20% mayor superando los demás tratamientos con 7,3 μg/ml. Estadísticamente no se encontraron diferencias significativas entre tratamientos (F=1,687; p=0,165; gl=5). El tratamiento con acetato de sodio 0,320 g/L + cloruro de sodio 1,28 g/L presentó el mayor crecimiento celular de 1,64x105 células/ml, presentándose diferencias significativas entre tratamientos (F=22,47; p=0,025 ; gl=5), y se observó para la concentración de clorofila (F=4,307; p=0,003; gl=5), obteniéndose para este tratamiento 0,245 μg/ml. La mayor producción de astaxantina se logró utilizando acetato de sodio desde el inicio del cultivo, seguido por la adición de cloruro de sodio al finalizar su fase exponencial. |
| publishDate |
2019 |
| dc.date.issued.none.fl_str_mv |
2019 |
| dc.date.accessioned.none.fl_str_mv |
2021-11-23T17:11:55Z |
| dc.date.available.none.fl_str_mv |
2021-11-23T17:11:55Z |
| dc.type.spa.fl_str_mv |
Trabajo de grado - Pregrado |
| dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_7a1f |
| dc.type.coarversion.spa.fl_str_mv |
http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/bachelorThesis |
| dc.type.redcol.spa.fl_str_mv |
https://purl.org/redcol/resource_type/TP |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
| format |
http://purl.org/coar/resource_type/c_7a1f |
| status_str |
publishedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://repositorio.unicolmayor.edu.co/handle/unicolmayor/3709 |
| dc.identifier.barcode.none.fl_str_mv |
60030 |
| url |
https://repositorio.unicolmayor.edu.co/handle/unicolmayor/3709 |
| identifier_str_mv |
60030 |
| dc.language.iso.spa.fl_str_mv |
spa |
| language |
spa |
| dc.relation.references.spa.fl_str_mv |
He B, Hou L, Dong M, Shi J, Huang X, Ding Y, Cong X, Zhang F, Zhang X, Zang X. Transcriptome Analysis in Haematococcus pluvialis: Astaxanthin Induction by High Light with Acetate and Fe2 . Int J Mol Sci. [internet] 2018;7(19) [consultado 2018 04 26] Pan-Utai W, Parakulksuksatid P, Phomkaivon N. Effect of inducing agents on growth and astaxanthin production in Haematococcus pluvialis: Organic and inorganic. Biocatalysis and Agricultural Biotechnology. [internet] 2017;12:152-158 [consultado 2018 04 26] Suyono E, Aminina , Pradania L, Mu’avatuna U , Habibaa R , Ramdaniyaha , Fatihatur E. Combination of blue, red, white, and ultraviolet lights for increasing carotenoids and biomass of Microalga Haematococcus pluvialis. Procedia Environmental Sciences. [internet] 2015; 28:399 – 405 [consultado 2018 04 26] Su Y, Wang J, Shi M, Niu X, Yu X, Gao L, Zhang X, Chen L, Zhang W. Metabolomic and network analysis of astaxanthin-producing Haematococcus pluvialis under various stress conditions. Bioresour Technol. [internet] 2014;170:522-529 [consultado 2018 04 26] Goksan T, Ak I, Gokpinar S. An alternative approach to the traditional mixotrophic cultures of Haematococcus pluvialis Flotow (Chlorophyceae). J Microbiol Biotechnol. [internet] 2010;20(9):1276-82. [consultado 2018 04 26] Vidhyavathi R, Venkatachalam L, Sarada R, Ravishankar GA. Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions. J Exp Bot. [internet] 2008;59(6):1409-18 [consultado 2018 04 26] Huang JC, Chen F, Sandmann G. Stress-related differential expression of multiple beta-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis. J Biotechnol. [internet] 2006;122(2):176-85 [consultado 2018 04 26] Jeon Y, Cho C, Yun Y. Combined effects of light intensity and acetate concentration on the growth of unicellular microalga Haematococcus pluvialis. Enzyme and Microbial Technology [internet] 2006;39(3):490-495 [consultado 2018 04 26] Orosa M, Franqueira D, Cid A, Abalde J. Analysis and enhancement of astaxanthin accumulation in Haematococcus pluvialis. Bioresour Technol. [internet] 2005;96(3):373-8. [consultado 2018 04 26] Wong Y, Ho Y, Ho K, Lai Y,Tsang P, Chow K, Yau Y, Choi M, Ho R. Effects of light intensity, illumination cycles on microalgae Haematococcus pluvialis for production of astaxanthin. Journal of marine biology and acquaculture. [internet] 2016;2(2):1-6. [consultado 2019 03 29] Wei D, Jing C, Zhao Y, Han B, Li T. Enhancing Haematococcus pluvialis biomass and aminobutyric acid accumulation by two-step cultivation and salt supplementation. Bioresour Technol. [internet] 2019; 285:121. [consultado 2019 06 09] Domínguez-Bocanegra AR, Guerrero Legarreta I, Martinez Jeronimo F, Tomasini Campocosio A. Influence of environmental and nutritional factors in the production of astaxanthin from Haematococcus pluvialis. Bioresour Technol. [internet] 2004;92(2):209-14. [consultado 2018 04 26] Feng L, Cai M, Li M, Huang X, Wang J. Accumulation of astaxanthin was improved by the nonmotile cells of Haematococcus pluvialis. Biomed Res Int [internet] 2019; 810:176. [consultado 2019 06 09] Cifuentes AS, González MA, Vargas S, Hoeneisen M, González N. Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions. Biol Res. [internet] 2003;36(3-4):343-57. [consultado 2018 04 26] Giannelli L, Yamada H, Katsuda T, Yamaji H. Effects of temperature on the astaxanthin productivity and light harvesting characteristics of the green alga Haematococcus pluvialis. Journal of Bioscience and bioengineering [internet] 2015;119(3):345-350 [consultado 2018 04 26] Sarada R, Bhattacharya S,Ravishankar G. Optimization of culture conditions for growth of the green alga Haematococcus pluvialis. World Journal of Microbiology and Biotechnology [internet] 2002; 18:517–521 [consultado 2018 04 26] Guedes C, Amaro H, Malcata F. Microalagae as sources of carotenoids. Mar Drugs [internet] 2011;625-644. [consultado 2018 03 10] Shah M, Liang Y, Cheng J, Daroch M. Astaxanthin-Producing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products. Front Plant Sci [internet] 2016;7(531) [consultado 2018 02 01] Cui H, Yu X, Wang Y, Cui Y, Li X. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae. BMC Genomics [internet] 2013;8(457) [consultado 2019 06 08] Galasso C, Corinaldesi C, Sansone C. Carotenoids from Marine Organisms: Biological Functions and Industrial Applications. Antioxidants [internet] 2017;6(4):96. [consultado 2018 02 01] Gong M, Bassi A. Carotenoids from microalgae: A review of recent developments. Biotechadv [internet] 2016;34(8):1396-1412 [consultado 2018 02 01] Davinelli S, Nielsen M, Scapagnini G. Astaxanthin in Skin Health, Repair, and Disease: A Comprehensive Review. Nutrients [internet] 2018;10(4):522 [consultado 2018 07 31] Shuehi O, Morita A, Ohnuki S, Hirata A, Sekida S, Okuda K, Ohya Y, Kawano S. Carotenoid dynamics and lipid droplet containing astaxanthin in response to light in the green alga Haematococcus pluvialis. Sci Rep [internet] 2018;8:5617 [consultado 2018 07 31] Wayama M, Ota Shuhei, Matsuura H, Nango N, Hirata A, Kawano S.Three- Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga Haematococcus pluvialis. PLoS One [internet] 2013;8(1):e53618 [consultado 2018 07 31] Hoang D, Tam L, Thuy N, Hong D. A study on the changes of the cell morphology, contents of pigments and intracellular protein in the life cycle of the green microalgal Haematococcus pluvialis under laboratory condition. Academia Journal of Biology. [internet] 2011;33(1) [consultado 2018 07 31] Gwak Y, Hwang Y, Wang B, Kim M, Jeong J, Lee C, Hu Q, Han D, Jin E. Comparative analyses of lipidomes and transcriptomes reveal a concerted action of multiple defensive systems against photooxidative stress in Haematococcus pluvialis. J. Exp. Bot. [internet] 2014;65(15):4317-4334 [consultado 2018 07 31] Imamoglu E, Sukan FV. Effect of Different Culture Media and Light Intensities on Growth of Haematococcus pluvialis. International journal of natural and engineering sciences [internet] 2007; 1(3):5–9 [consultado 2018 08 04] Niño CM, Rodríguez FC, Díaz LE, Lancheros AG. Evaluación de las condiciones de crecimiento celular para la producción de astaxantina a patir de la microalga Haematococcus pluvialis. NOVA. [internet] 2017;15(28) [consultado 2018 08 04] Li Y, Sommerfeld M, Chen F, Hu Q. Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). J Plant Physiol. [internet] 2008;165(17):1783-97 [consultado 2018 08 20] Christian D, Zhang J, Sawdown A, Pen C. Enhanced astaxanthin accumulation in Haematococcus pluvialis using high carbon dioxide concentration and light illumination. Bioresour Technol [internet] 2018; 256:548-551 [consultado 2018 08 20] Sheng B, Fan F, Huang J, Bai W, Wang J, Li S, Li W, Wan M, Li Y. Investigation on models for light distribution of Haematococcus pluvialis during astaxanthin accumulation stage with an application case. Algal research [internet] 2018; 33:182-189 [consultado 2018 08 20] Kim Z, Kim S, Lee H, Lee C. Enhanced production of astaxanthin by flashing light using Haematococcus pluvialis. Enzyme and Microbial Technology [internet] 2006; 39:414-419 [consultado 2018 08 20] Borowitzka M., Beardall J., Raven J. The physiology of microalgae.1 ed. [internet]. Suiza: Springer-Verlag GmbH; 2016 [consultado 2018 08 23]. Richmond A. Handbook of microalgal Mass culture. Segunda edición. Inglaterra: Wiley Balckwell; 2013. Jin H, Lao YM, Zhou J, Zhang HJ, Cai ZH. Simultaneous determination of 13 carotenoids by a simple C18 column-based ultra-high-pressure liquid chromatography method for carotenoid profiling in the astaxanthin- accumulating Haematococcus pluvialis. J Chromatogr A. [internet] 2017; 10:93- 103 [consultado 2018 09 16] Steinbrenner J, Sandmann G. Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin byosinthesis. Appl Enviroment Microbiol. [internet] 2006;72(12):7477-84 [consultado 2018 09 16] Arredondo B, Voltolina D. Concentración, recuento celular y tasa de crecimiento. Métodos y Herramientas Analíticas en la Evaluación de la Biomasa Microalgal. [internet] 2014 [consultado 2018 09 16] Harker M, Tsavalos A, Young A. Autotrophic growth and carotenoid production of Haematococcus pluvialis in a 30 liter air-lift photobioreactor. J Ferment Bioeng. [internet] 1996;82: 101-106 [consultado 2019 02 07] Harker M, Tsavalos A, Young A. Factors responsible for astaxanthin formation in the chlorophyte Haematococcus pluvialis. Bioresourse Technology. [internet] 1995;55:207-241 [consultado 2019 02 07] Sarada R, Tripathi U, Ravishankar G. Influence of stress on astaxanthin production in Haematococcus pluvialis grown under different culture conditions. Process Biochemistry. [internet] 2002;37:623–627 [consultado 2019 02 07] Gao F, Yang H, Li C, Pen Y, Lu M. Effect of organic carbon to nitrogen ratio in wastewater on growth, nutrient uptake and lipid accumulation of a mixotrophic microalgae Chlorella sp. Bioresour technol. [internet] 2019;282:118- 224 [consultado 2019 06 09] Wan M, Zhang J, Hou D, Fan J, Li Y, Huang J, Wang J. The effect of temperature on cell growth and astaxanthin accumulation of Haematococcus pluvialis during a light-dark cyclic cultivation. Bioresour Technol. [internet] 2014;167:276-283 [consultado 2018 08 11] Chojnacka K, Marquez F. Kinetic and Stoichiometric Relationships of the Energy and Carbon Metabolism in the Culture of Microalgae. Biotechnol. [internet] 2004;3(1):21-34 [consultado 2019 03 22] Wayne K, Reen S, Loke P, Jiun Y, Chuan T, Chang J. Effects of water culture medium, cultivation systems and growth modes for microalgae cultivation: A review. Journal of the Taiwan Institute of Chemical Engineers. [internet] 2018;91:332–344 [consultado 2019 03 22] Li Q, Zhang L, Liu J. Comparative transcriptome analysis at seven time points during Haematococcus pluvialismotile cell growth and astaxanthin accumulation. Aquaculture. [internet] 2019;503:304-311 [consultado 2019 03 22] Zhang C, Liu J, Zhang L. Cell cycles and proliferation patterns in Haematococcus pluvialis. Chinese Journal of Oceanology and Limnology. [internet] 2017;35(5):1205-1211 [consultado 2019 03 22] Li Y, Sommerfeld M, Chen F, Hu Q. Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae). J Appl Phycol.. [internet] 2010;22(3):253-263 [consultado 2019 03 22] Morais M, Silva B, Greque E, Vieira J. Biologically active metabolites synthesized by microalgae. Biomed Research International. [internet] 2015;15 [consultado 2019 06 08] Gwozdz T, Dorey K. Basic Science Methods for Clinical Researchers. Reino Unido: Academic Press;2017. Weston A, Brown P. HPLC and CE Principles and Practice. Estados Unidos: Academic Press;1997. Cai M, Li F. Recent advances in Haematococcus pluvialis scale culture technology. Natural Science. [internet] 2016;55(5):733-741 [consultado 2019 06 09] Guerin M, Huntley M, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends in Biotechnology. [internet] 2003;21(5):210- 216 [consultado 2019 03 28] Gu W, Xie X, Gao S, Zhou W, Pan G, Wang G. Comparison of Different Cells of Haematococcus pluvialis Reveals an Extensive Acclimation Mechanism during its Aging Process: From a Perspective of Photosynthesis. PLoS One. [internet] 2013;8(7): e67028. [consultado 2019 03 28] Olaizola M. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomolecular Engineering. [internet] 2003;20(4):459-466 [consultado 2019 03 28] Higuera I, Valenzuela F,Goycoolea F. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr. [internet] 2006;46(2):185-96. [consultado 2019 03 28] Chekanov K, Lukyanov A, Boussiba S, Aflalo C, Solovchenko A. Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back.Photosynthesis Research. [internet] 2016;128(3):313-323. [consultado 2019 03 28] Pelt-Verkuil E, Belkum A, Hays J. Principals and technical aspects of PCR amplification. Holanda: Springer;2008 Shi J, Zang X, Cong X, Hou L, He B, Ding Y, Dong M, Sun D, Guo Y, Zhang F, Wang Z, Wei X, Zhang X, . Cloning of nitrite reductase gene from Haematococcus pluvialis and transcription and enzymatic activity analysis at different nitrate and phosphorus concentration. Gene. [internet] 2019;697:123-130. [consultado 2019 03 28] Tang Z, Zhu S, Fei L, Wei L, Xue S. Effects of Astaxanthin on Reverse Cholesterol Transport and Atherosclerosis in Mice. Biomed Res Int [internet] 2017;25:932. [consultado 2019 06 09] Kakizono T, Kobayashi M, Nagai S. Effect of carbon/nitrogen ratio on encystment accompanied with astaxanthin formation in a green alga, Haematococcus pluvialis. Journal of Fermentation and Bioengineering. [internet] 1992;74(6):403- 405. [consultado 2019 03 29] Hong M.-E., Hwang S. K., Chang W. S., Kim B. W., Lee J., Sim S. J. Enhanced autotrophic astaxanthin production from Haematococcus pluvialis under high temperature via heat stress-driven Haber–Weiss reaction. Applied Microbiology and Biotechnology. [internet] 2015;99(12):5203–5215 [consultado 2019 06 09] Wan M, Zhang J, Hou D, Fan J, Li Y, Huang J, Wang J. The effect of temperature on cell growth and astaxanthin accumulation of Haematococcus pluvialis during a light–dark cyclic cultivation. Bioresour Technol [internet] 2014;167:276–283 [consultado 2019 06 09] Nguyen K. Astaxanthin: a comparative case of synthetic VS Natural production. Chemical and Biomolecular Engineering. [internet] 2013. [consultado 2019 04 01] Capelli B, Bagchi D, Cysewski G . Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutraceutical supplement. Nutrafoods. [internet] 2013;12(4):145-152. [consultado 2019 04 01] Bai J, Beena B, Shashirekha V. Nomenclature,Taxonomy, Reproduction and Life Cycle of the genus Haematococcus, Haematococcaceae, Chlorophycea. Phykos. [internet] 2016;46(1):64-70. [consultado 2019 04 02] Peled E, Pick U, Zarka A, Shimoni E, Leu S, Boussiba S. Light-induced oil globule migration in haematococcus pluvialis (chlorophyceae). J Phycol. [internet] 2012;48(5):1209-1219. [consultado 2019 04 02] Lemoine Y, Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res. [internet] 2010 Nov;106(1- 2):155-77. [consultado 2019 04 02] Shen Y., Cai M. G., Huang S. Y., et al. Haematococcus pluvialis culture in photobioreactor. Marine Sciences. [internet] 2010;34(10):83–89 . [consultado 2019 06 09] Sun H., Liu B., Lu X., Cheng K.-W., Chen F. Staged cultivation enhances biomass accumulation in the green growth phase of Haematococcus pluvialis. Bioresource Technology. [internet] 2017;233:326–331. [consultado 2019 06 09] Brinda BR, Sarada R, Kamath BS, Ravishankar GA. Accumulation of astaxanthin in flagellated cells of Haematococcus pluvialis – cultural and regulatory aspects. Curr Sci. [internet] 2004;87:1290–1295 [consultado 2019 04 03] Wang J., Han D., Sommerfeld M. R., Lu C., Hu Q. Effect of initial biomass density on growth and astaxanthin production of Haematococcus pluvialis in an outdoor photobioreactor. Journal of Applied Phycology. [internet] 2013;25(1):253–260 [consultado 2019 06 09] Li Y., Sommerfeld M., Chen F., Hu Q. Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae) Journal of Applied Phycology. [internet] 2010;22(3):253–263 [consultado 2019 06 09] Luo Q, Bian C, Tao M, Huang Y, Lv Y. Genome and Transcriptome Sequencing of the Astaxanthin-Producing Green Microalga, Haematococcus pluvialis. Genome Biol Evol.. [internet] 2019;11(1):166-173 [consultado 2019 06 09] Tufan M, Sayin S. The effects of iron and light intensity on biomass and pigment synthesis of heamotococcus pluvialis under laboratory conditions. ICAMS. [internet] 2014 [consultado 2019 04 03] Kobayashi M, Kakizono T, Nishio N, Nagai S. Effects of light intensity, light quality, and illumination cycle on astaxanthin formation in a green alga, Haematococcus pluvialis. Journal of Fermentation and Bioengineering. [internet] 1992;74(1):61-63 [consultado 2019 04 03] Kobayashi M, Kakizono T, Nagai S. Enhanced Carotenoid Biosynthesis by Oxidative Stress in Acetate-Induced Cyst Cells of a Green Unicellular Alga, Haematococcus pluvialis. Appl Environ Microbiol. [internet] 1993;59(3):867-73. [consultado 2019 04 03] Kobayashi M, Kurimura Y, Kakizono T, Nishio N, Tsuji Y. Morphological changes in the life cycle of the green alga Haematococcus pluvialis. Journal of Fermentation and Bioengineering. [internet] 1997;84(1):94-97 [consultado 2019 04 03] Li Y, Huang J, Sandmann G, Chen F. High‐light and sodium chloride stress differentially regulate the biosynthesis of astaxanthin in Chlorella zofingiensis (Chlorophyceae). Journal of Phycology. [internet] 2009;45(3) [consultado 2019 04 03] Meng CX, Teng CY, Jiang P, Qin S, Tseng C. Cloning and characterization of β- carotene ketolase gene promoter in Haematococcus pluvialis. Acta Biochim Biophys Sinica [internet] 2005; 37:270–275 [consultado 2019 04 03] Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends in plant science. [internet] 2002;7(9):405-410 [consultado 2019 04 03] Olaizola M, Huntley ME. Recent advances in commercial production of astaxanthin from microalgae. Recent advances in marine biotechnology. [internet] 2003;9:143–164 Orosa M, Valero J, Herrero C, Abalde J. Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N- starvation and high light conditions. Biotechnology Letters. [internet] 2001;23(13):1079–1085 [consultado 2019 04 03] Park EK, Lee CG. Astaxanthin production by Haematococcus pluvialis under various light intensities and wavelengths. J Microbiol Biotechnol. [internet] 2001; 11:1024–1030 [consultado 2019 04 03] Pelah D, Sintov A, Cohen E. The effect of salt stress on the production of canthaxanthin and astaxanthin by Chlorella zofingiensis grown under limited light intensity. World J Microbiol Biotechnol. [internet] 2004; 20:483–486 [consultado 2019 04 03] Qiu B, Li Y. Photosynthetic acclimation and photoprotective mechanism in Haematococcus pluvialis (Chlorophyceae) during the accumulation of secondary carotenoids at elevated irradiation. Phycologia. [internet] 2006; 45:117–126 [consultado 2019 04 03] Khan Academy [internet] Estados Unidos; c2006. [consultado 2019 04 03]. Disponible en: https://es.khanacademy.org/science/biology/photosynthesis-in- plants/the-light-dependent-reactions-of-photosynthesis/a/light-dependent- reactions Saini RK, Keum Y. Progress in microbial carotenoids production. Indian J Microbiol [internet] 2017;57:129–130 [consultado 2019 06 09] Mascia F, Girolomoni L, Alcocer M, Bargigia I, Perozeni F, Cazzaniga S, Cerullo G. Functional analysis of photosynthetic pigment binding complexes in the green alga Haematococcus pluvialis reveals distribution of astaxanthin in Photosystems. Sci Rep. [internet] 2017; 7: 16319. [consultado 2019 04 03] Bhosale P, Bernstein P. Microbial xanthophylls. Appl Microbiol Biotechnol. . [internet] 2015;68:445–455 [consultado 2019 06 09] Steinbrenner J, Linden H. Regulation of two carotenoid biosynthetic genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiol. [internet] 2001; 125:810–817. [consultado 2019 04 03] Steinbrenner J, Linden H (2003) Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol. [internet] 2003; 52:343–356. [consultado 2019 04 03] Tan S, Cunningham FX, Youmans M, Grabowski B, Sun Z, Gantt E. Cytochrome f loss in astaxanthin-accumulating red cells of Haematococcus pluvialis (Chlorophyceae): comparison of photosynthetic activity, photosynthetic enzymes, and thylakoid membrane polypeptides in red and green cells. J Phycol. [internet] 1995; 31:897–905. [consultado 2019 04 03] Tran N-P, Park J-K, Lee C-G. Proteomic analysis of proteins in green alga Haematococcus pluvialis (Chlorophyceae) expressed under combined stress of nitrogen starvation and high irradiance. Enzyme Microb Technol. [internet] 2009; 45:241–246 [consultado 2019 04 03] Vidhyavathi R, Sarada R, Rhavishankar GA. Expression of carotenogenic genes and carotenoid production in Haematococcus pluvialis under the influence of carotenoid and fatty acid synthesis inhibitors. Enzyme Microb Technol. [internet] 2009; 45:88–93 [consultado 2019 04 03] Wang Y, Peng J. Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta). World J Microbiol Biotechnol. [internet] 2008; 24:1915–1922 [consultado 2019 04 03] Wang B, Zarka A, Trebst A, Boussiba S. Astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae) as an active photoprotective process during high irradiance. J Phycol. [internet] 2008; 39:1116–1124 [consultado 2019 04 03] |
| dc.rights.spa.fl_str_mv |
Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2019 |
| dc.rights.uri.spa.fl_str_mv |
https://creativecommons.org/licenses/by-nc-sa/4.0/ |
| dc.rights.accessrights.spa.fl_str_mv |
info:eu-repo/semantics/closedAccess |
| dc.rights.creativecommons.spa.fl_str_mv |
Atribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0) |
| dc.rights.coar.spa.fl_str_mv |
http://purl.org/coar/access_right/c_14cb |
| rights_invalid_str_mv |
Derechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2019 https://creativecommons.org/licenses/by-nc-sa/4.0/ Atribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0) http://purl.org/coar/access_right/c_14cb |
| eu_rights_str_mv |
closedAccess |
| dc.format.extent.spa.fl_str_mv |
100p. |
| dc.format.mimetype.spa.fl_str_mv |
application/pdf |
| dc.publisher.spa.fl_str_mv |
Universidad Colegio Mayor de Cundinamarca |
| dc.publisher.faculty.spa.fl_str_mv |
Facultad de Ciencias de la Salud |
| dc.publisher.place.spa.fl_str_mv |
Bogotá |
| dc.publisher.program.spa.fl_str_mv |
Bacteriología y Laboratorio Clínico |
| institution |
Colegio Mayor de Cundinamarca |
| bitstream.url.fl_str_mv |
https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/1/PRESENTACION.pdf https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/2/IDENTIFICACION%20TRABAJO%20DE%20GRADO.pdf https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/3/RODRIGUEZ%20ROMERO%20LAURA%20JOHANNA-.pdf https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/4/license.txt https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/5/PRESENTACION.pdf.txt https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/7/IDENTIFICACION%20TRABAJO%20DE%20GRADO.pdf.txt https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/9/RODRIGUEZ%20ROMERO%20LAURA%20JOHANNA-.pdf.txt https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/6/PRESENTACION.pdf.jpg https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/8/IDENTIFICACION%20TRABAJO%20DE%20GRADO.pdf.jpg https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/10/RODRIGUEZ%20ROMERO%20LAURA%20JOHANNA-.pdf.jpg |
| bitstream.checksum.fl_str_mv |
2ed531e689c6cd97c3c14ee272c91d7a 41ceb11ab802d4837c1d58fb598b747f 562b3ac7b65f0c69c5145adaf8179408 2f9959eaf5b71fae44bbf9ec84150c7a bbb6f7c592cdad8d1ba91c6ba028b87d 14857a64d494d499c8ea946e1585dd67 e928b6577dd1ebbc27e785b9062b628a c7173fd47663ed735410e8433392e006 7390c348849db7558b291e1adef7e63e d2c143b1502cc5812ca08afc0a7de007 |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 MD5 MD5 MD5 MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Biblioteca Digital Unicolmayor |
| repository.mail.fl_str_mv |
repositorio@unicolmayor.edu.co |
| _version_ |
1812210113531871232 |
| spelling |
Camacho Kurmen, Judith Elena88ec3d87924bea44fb175e7780652267Rodríguez Romero, Laura Johannaa80869bf8933de5a75fb4db1358745ef2021-11-23T17:11:55Z2021-11-23T17:11:55Z2019https://repositorio.unicolmayor.edu.co/handle/unicolmayor/370960030H. pluvialis es una microalga utilizada para la producción de astaxantina, este es un betacaroteno y antioxidante en la industria. El objetivo del trabajo es utilizar diferentes factores de estrés para obtener una mayor producción de astaxantina, la microalga se cultivó en medio RM bajo condiciones de pH 6,8, temperatura 20±2oC, aire filtrado, iluminación con lámparas blancas con 20h luz/4h oscuridad, de 70 μE m−2 s −1 con diferentes concentraciones de acetato de sodio y cloruro de sodio adicionadas a diferentes tiempos. Este estudio se realizó durante 56 días. Se determinó el crecimiento celular, el análisis de los cambios morfológicos y la cuantificación de astaxantina y clorofila por el método de espectrofotometría. Se realizó un análisis estadístico utilizando ANOVA (95%). Se evidencio una mayor producción de astaxantina al ensayo adicionado con 1,6 g/L de acetato de sodio y 6,4 g/L de cloruro de sodio. Un 20% mayor superando los demás tratamientos con 7,3 μg/ml. Estadísticamente no se encontraron diferencias significativas entre tratamientos (F=1,687; p=0,165; gl=5). El tratamiento con acetato de sodio 0,320 g/L + cloruro de sodio 1,28 g/L presentó el mayor crecimiento celular de 1,64x105 células/ml, presentándose diferencias significativas entre tratamientos (F=22,47; p=0,025 ; gl=5), y se observó para la concentración de clorofila (F=4,307; p=0,003; gl=5), obteniéndose para este tratamiento 0,245 μg/ml. La mayor producción de astaxantina se logró utilizando acetato de sodio desde el inicio del cultivo, seguido por la adición de cloruro de sodio al finalizar su fase exponencial.Resumen 1 Introducción 2 Objetivos 4 1. Antecedentes 5 2. Marco referencial 16 2.1 Haematococcus pluvialis 16 2.1.1 Taxonomía y localización 16 2.1.2 Ciclo biológico 17 2.1.3 Usos 20 2.1.4 Cultivo de H. pluvialis para la producción de astaxantina 20 2.2 Astaxantina 21 2.2.1 Usos de astaxantina 23 2.2.2 Ruta metabólica para producir astaxantina 24 2.3 Clorofila 27 2.4 Factores que promueven el crecimiento de H. pluvialis 31 2.4.1 Cultivos 31 2.4.1.1 Cultivo fotoautotrófico 31 2.4.1.2 Cultivo mixotrofico 32 2.4.2 Luz 33 2.4.3 Temperatura 33 2.4.4 Nitrógeno 34 2.4.5 Fosforo 35 2.4.6 Hierro 36 2.5 Factores que promueven la producción de astaxantina y clorofila en H. pluvialis 36 2.5.1 Luz 36 2.5.2 Sales 38 2.6 Métodos de medición de biomasa 39 2.6.1 Cámara de Neubauer 39 2.6.2 Peso seco 40 2.6.3 Densidad óptica 40 2.6.4 Epifluorescencia 41 2.7 Métodos de medición de astaxantina y clorofila 41 2.7.1 Espectrofotómetro 41 2.7.2 HPLC 41 2.8 Métodos de medición de proteínas 42 2.8.1 Electroforesis 42 2.8.2 Western blot 43 2.8.3 PCR 44 3. Diseño metodológico 45 3.1 Universo población, muestra 45 3.2 Hipótesis, variables e indicadores 45 3.3 Técnicas y procedimientos 46 3.3.1 Fase 1 Crecimiento de Haematococcus pluvialis 46 3.3.2 Fase 2 Cambios morfológicos celulares 47 3.3.3 Fase 3 Cuantificación de astaxantina y clorofila 47 4. Resultados 48 4.1 FASE 1 Evaluación crecimiento de H. pluvialis 48 4.2 FASE 2 Evaluación cambios morfológicos macroscópicos y microscópicos de H. pluvialis 53 4.3 FASE 3 Cuantificación astaxantina y clorofila 61 5. Discusión 66 6. Conclusiones 72 Recomendaciones 74 Referencias bibliográficas 75 Anexos 85 Agradecimientos 90PregradoBacteriólogo(a) y Laboratorista Clínico100p.application/pdfspaUniversidad Colegio Mayor de CundinamarcaFacultad de Ciencias de la SaludBogotáBacteriología y Laboratorio ClínicoDerechos Reservados - Universidad Colegio Mayor de Cundinamarca, 2019https://creativecommons.org/licenses/by-nc-sa/4.0/info:eu-repo/semantics/closedAccessAtribución-NoComercial-CompartirIgual 4.0 Internacional (CC BY-NC-SA 4.0)http://purl.org/coar/access_right/c_14cbProducción de astaxantina en Haematococcus pluvialis bajo efecto de factores de estrés como acetato de sodio y cloruro de sodioTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fhttp://purl.org/coar/version/c_970fb48d4fbd8a85Textinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPinfo:eu-repo/semantics/publishedVersionHe B, Hou L, Dong M, Shi J, Huang X, Ding Y, Cong X, Zhang F, Zhang X, Zang X. Transcriptome Analysis in Haematococcus pluvialis: Astaxanthin Induction by High Light with Acetate and Fe2 . Int J Mol Sci. [internet] 2018;7(19) [consultado 2018 04 26]Pan-Utai W, Parakulksuksatid P, Phomkaivon N. Effect of inducing agents on growth and astaxanthin production in Haematococcus pluvialis: Organic and inorganic. Biocatalysis and Agricultural Biotechnology. [internet] 2017;12:152-158 [consultado 2018 04 26]Suyono E, Aminina , Pradania L, Mu’avatuna U , Habibaa R , Ramdaniyaha , Fatihatur E. Combination of blue, red, white, and ultraviolet lights for increasing carotenoids and biomass of Microalga Haematococcus pluvialis. Procedia Environmental Sciences. [internet] 2015; 28:399 – 405 [consultado 2018 04 26]Su Y, Wang J, Shi M, Niu X, Yu X, Gao L, Zhang X, Chen L, Zhang W. Metabolomic and network analysis of astaxanthin-producing Haematococcus pluvialis under various stress conditions. Bioresour Technol. [internet] 2014;170:522-529 [consultado 2018 04 26]Goksan T, Ak I, Gokpinar S. An alternative approach to the traditional mixotrophic cultures of Haematococcus pluvialis Flotow (Chlorophyceae). J Microbiol Biotechnol. [internet] 2010;20(9):1276-82. [consultado 2018 04 26]Vidhyavathi R, Venkatachalam L, Sarada R, Ravishankar GA. Regulation of carotenoid biosynthetic genes expression and carotenoid accumulation in the green alga Haematococcus pluvialis under nutrient stress conditions. J Exp Bot. [internet] 2008;59(6):1409-18 [consultado 2018 04 26]Huang JC, Chen F, Sandmann G. Stress-related differential expression of multiple beta-carotene ketolase genes in the unicellular green alga Haematococcus pluvialis. J Biotechnol. [internet] 2006;122(2):176-85 [consultado 2018 04 26]Jeon Y, Cho C, Yun Y. Combined effects of light intensity and acetate concentration on the growth of unicellular microalga Haematococcus pluvialis. Enzyme and Microbial Technology [internet] 2006;39(3):490-495 [consultado 2018 04 26]Orosa M, Franqueira D, Cid A, Abalde J. Analysis and enhancement of astaxanthin accumulation in Haematococcus pluvialis. Bioresour Technol. [internet] 2005;96(3):373-8. [consultado 2018 04 26]Wong Y, Ho Y, Ho K, Lai Y,Tsang P, Chow K, Yau Y, Choi M, Ho R. Effects of light intensity, illumination cycles on microalgae Haematococcus pluvialis for production of astaxanthin. Journal of marine biology and acquaculture. [internet] 2016;2(2):1-6. [consultado 2019 03 29]Wei D, Jing C, Zhao Y, Han B, Li T. Enhancing Haematococcus pluvialis biomass and aminobutyric acid accumulation by two-step cultivation and salt supplementation. Bioresour Technol. [internet] 2019; 285:121. [consultado 2019 06 09]Domínguez-Bocanegra AR, Guerrero Legarreta I, Martinez Jeronimo F, Tomasini Campocosio A. Influence of environmental and nutritional factors in the production of astaxanthin from Haematococcus pluvialis. Bioresour Technol. [internet] 2004;92(2):209-14. [consultado 2018 04 26]Feng L, Cai M, Li M, Huang X, Wang J. Accumulation of astaxanthin was improved by the nonmotile cells of Haematococcus pluvialis. Biomed Res Int [internet] 2019; 810:176. [consultado 2019 06 09]Cifuentes AS, González MA, Vargas S, Hoeneisen M, González N. Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions. Biol Res. [internet] 2003;36(3-4):343-57. [consultado 2018 04 26]Giannelli L, Yamada H, Katsuda T, Yamaji H. Effects of temperature on the astaxanthin productivity and light harvesting characteristics of the green alga Haematococcus pluvialis. Journal of Bioscience and bioengineering [internet] 2015;119(3):345-350 [consultado 2018 04 26]Sarada R, Bhattacharya S,Ravishankar G. Optimization of culture conditions for growth of the green alga Haematococcus pluvialis. World Journal of Microbiology and Biotechnology [internet] 2002; 18:517–521 [consultado 2018 04 26]Guedes C, Amaro H, Malcata F. Microalagae as sources of carotenoids. Mar Drugs [internet] 2011;625-644. [consultado 2018 03 10]Shah M, Liang Y, Cheng J, Daroch M. Astaxanthin-Producing Green Microalga Haematococcus pluvialis: From Single Cell to High Value Commercial Products. Front Plant Sci [internet] 2016;7(531) [consultado 2018 02 01]Cui H, Yu X, Wang Y, Cui Y, Li X. Evolutionary origins, molecular cloning and expression of carotenoid hydroxylases in eukaryotic photosynthetic algae. BMC Genomics [internet] 2013;8(457) [consultado 2019 06 08]Galasso C, Corinaldesi C, Sansone C. Carotenoids from Marine Organisms: Biological Functions and Industrial Applications. Antioxidants [internet] 2017;6(4):96. [consultado 2018 02 01]Gong M, Bassi A. Carotenoids from microalgae: A review of recent developments. Biotechadv [internet] 2016;34(8):1396-1412 [consultado 2018 02 01]Davinelli S, Nielsen M, Scapagnini G. Astaxanthin in Skin Health, Repair, and Disease: A Comprehensive Review. Nutrients [internet] 2018;10(4):522 [consultado 2018 07 31]Shuehi O, Morita A, Ohnuki S, Hirata A, Sekida S, Okuda K, Ohya Y, Kawano S. Carotenoid dynamics and lipid droplet containing astaxanthin in response to light in the green alga Haematococcus pluvialis. Sci Rep [internet] 2018;8:5617 [consultado 2018 07 31]Wayama M, Ota Shuhei, Matsuura H, Nango N, Hirata A, Kawano S.Three- Dimensional Ultrastructural Study of Oil and Astaxanthin Accumulation during Encystment in the Green Alga Haematococcus pluvialis. PLoS One [internet] 2013;8(1):e53618 [consultado 2018 07 31]Hoang D, Tam L, Thuy N, Hong D. A study on the changes of the cell morphology, contents of pigments and intracellular protein in the life cycle of the green microalgal Haematococcus pluvialis under laboratory condition. Academia Journal of Biology. [internet] 2011;33(1) [consultado 2018 07 31]Gwak Y, Hwang Y, Wang B, Kim M, Jeong J, Lee C, Hu Q, Han D, Jin E. Comparative analyses of lipidomes and transcriptomes reveal a concerted action of multiple defensive systems against photooxidative stress in Haematococcus pluvialis. J. Exp. Bot. [internet] 2014;65(15):4317-4334 [consultado 2018 07 31]Imamoglu E, Sukan FV. Effect of Different Culture Media and Light Intensities on Growth of Haematococcus pluvialis. International journal of natural and engineering sciences [internet] 2007; 1(3):5–9 [consultado 2018 08 04]Niño CM, Rodríguez FC, Díaz LE, Lancheros AG. Evaluación de las condiciones de crecimiento celular para la producción de astaxantina a patir de la microalga Haematococcus pluvialis. NOVA. [internet] 2017;15(28) [consultado 2018 08 04]Li Y, Sommerfeld M, Chen F, Hu Q. Consumption of oxygen by astaxanthin biosynthesis: a protective mechanism against oxidative stress in Haematococcus pluvialis (Chlorophyceae). J Plant Physiol. [internet] 2008;165(17):1783-97 [consultado 2018 08 20]Christian D, Zhang J, Sawdown A, Pen C. Enhanced astaxanthin accumulation in Haematococcus pluvialis using high carbon dioxide concentration and light illumination. Bioresour Technol [internet] 2018; 256:548-551 [consultado 2018 08 20]Sheng B, Fan F, Huang J, Bai W, Wang J, Li S, Li W, Wan M, Li Y. Investigation on models for light distribution of Haematococcus pluvialis during astaxanthin accumulation stage with an application case. Algal research [internet] 2018; 33:182-189 [consultado 2018 08 20]Kim Z, Kim S, Lee H, Lee C. Enhanced production of astaxanthin by flashing light using Haematococcus pluvialis. Enzyme and Microbial Technology [internet] 2006; 39:414-419 [consultado 2018 08 20]Borowitzka M., Beardall J., Raven J. The physiology of microalgae.1 ed. [internet]. Suiza: Springer-Verlag GmbH; 2016 [consultado 2018 08 23].Richmond A. Handbook of microalgal Mass culture. Segunda edición. Inglaterra: Wiley Balckwell; 2013.Jin H, Lao YM, Zhou J, Zhang HJ, Cai ZH. Simultaneous determination of 13 carotenoids by a simple C18 column-based ultra-high-pressure liquid chromatography method for carotenoid profiling in the astaxanthin- accumulating Haematococcus pluvialis. J Chromatogr A. [internet] 2017; 10:93- 103 [consultado 2018 09 16]Steinbrenner J, Sandmann G. Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin byosinthesis. Appl Enviroment Microbiol. [internet] 2006;72(12):7477-84 [consultado 2018 09 16]Arredondo B, Voltolina D. Concentración, recuento celular y tasa de crecimiento. Métodos y Herramientas Analíticas en la Evaluación de la Biomasa Microalgal. [internet] 2014 [consultado 2018 09 16]Harker M, Tsavalos A, Young A. Autotrophic growth and carotenoid production of Haematococcus pluvialis in a 30 liter air-lift photobioreactor. J Ferment Bioeng. [internet] 1996;82: 101-106 [consultado 2019 02 07]Harker M, Tsavalos A, Young A. Factors responsible for astaxanthin formation in the chlorophyte Haematococcus pluvialis. Bioresourse Technology. [internet] 1995;55:207-241 [consultado 2019 02 07]Sarada R, Tripathi U, Ravishankar G. Influence of stress on astaxanthin production in Haematococcus pluvialis grown under different culture conditions. Process Biochemistry. [internet] 2002;37:623–627 [consultado 2019 02 07]Gao F, Yang H, Li C, Pen Y, Lu M. Effect of organic carbon to nitrogen ratio in wastewater on growth, nutrient uptake and lipid accumulation of a mixotrophic microalgae Chlorella sp. Bioresour technol. [internet] 2019;282:118- 224 [consultado 2019 06 09]Wan M, Zhang J, Hou D, Fan J, Li Y, Huang J, Wang J. The effect of temperature on cell growth and astaxanthin accumulation of Haematococcus pluvialis during a light-dark cyclic cultivation. Bioresour Technol. [internet] 2014;167:276-283 [consultado 2018 08 11]Chojnacka K, Marquez F. Kinetic and Stoichiometric Relationships of the Energy and Carbon Metabolism in the Culture of Microalgae. Biotechnol. [internet] 2004;3(1):21-34 [consultado 2019 03 22]Wayne K, Reen S, Loke P, Jiun Y, Chuan T, Chang J. Effects of water culture medium, cultivation systems and growth modes for microalgae cultivation: A review. Journal of the Taiwan Institute of Chemical Engineers. [internet] 2018;91:332–344 [consultado 2019 03 22]Li Q, Zhang L, Liu J. Comparative transcriptome analysis at seven time points during Haematococcus pluvialismotile cell growth and astaxanthin accumulation. Aquaculture. [internet] 2019;503:304-311 [consultado 2019 03 22]Zhang C, Liu J, Zhang L. Cell cycles and proliferation patterns in Haematococcus pluvialis. Chinese Journal of Oceanology and Limnology. [internet] 2017;35(5):1205-1211 [consultado 2019 03 22]Li Y, Sommerfeld M, Chen F, Hu Q. Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae). J Appl Phycol.. [internet] 2010;22(3):253-263 [consultado 2019 03 22]Morais M, Silva B, Greque E, Vieira J. Biologically active metabolites synthesized by microalgae. Biomed Research International. [internet] 2015;15 [consultado 2019 06 08]Gwozdz T, Dorey K. Basic Science Methods for Clinical Researchers. Reino Unido: Academic Press;2017.Weston A, Brown P. HPLC and CE Principles and Practice. Estados Unidos: Academic Press;1997.Cai M, Li F. Recent advances in Haematococcus pluvialis scale culture technology. Natural Science. [internet] 2016;55(5):733-741 [consultado 2019 06 09]Guerin M, Huntley M, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends in Biotechnology. [internet] 2003;21(5):210- 216 [consultado 2019 03 28]Gu W, Xie X, Gao S, Zhou W, Pan G, Wang G. Comparison of Different Cells of Haematococcus pluvialis Reveals an Extensive Acclimation Mechanism during its Aging Process: From a Perspective of Photosynthesis. PLoS One. [internet] 2013;8(7): e67028. [consultado 2019 03 28]Olaizola M. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomolecular Engineering. [internet] 2003;20(4):459-466 [consultado 2019 03 28]Higuera I, Valenzuela F,Goycoolea F. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr. [internet] 2006;46(2):185-96. [consultado 2019 03 28]Chekanov K, Lukyanov A, Boussiba S, Aflalo C, Solovchenko A. Modulation of photosynthetic activity and photoprotection in Haematococcus pluvialis cells during their conversion into haematocysts and back.Photosynthesis Research. [internet] 2016;128(3):313-323. [consultado 2019 03 28]Pelt-Verkuil E, Belkum A, Hays J. Principals and technical aspects of PCR amplification. Holanda: Springer;2008Shi J, Zang X, Cong X, Hou L, He B, Ding Y, Dong M, Sun D, Guo Y, Zhang F, Wang Z, Wei X, Zhang X, . Cloning of nitrite reductase gene from Haematococcus pluvialis and transcription and enzymatic activity analysis at different nitrate and phosphorus concentration. Gene. [internet] 2019;697:123-130. [consultado 2019 03 28]Tang Z, Zhu S, Fei L, Wei L, Xue S. Effects of Astaxanthin on Reverse Cholesterol Transport and Atherosclerosis in Mice. Biomed Res Int [internet] 2017;25:932. [consultado 2019 06 09]Kakizono T, Kobayashi M, Nagai S. Effect of carbon/nitrogen ratio on encystment accompanied with astaxanthin formation in a green alga, Haematococcus pluvialis. Journal of Fermentation and Bioengineering. [internet] 1992;74(6):403- 405. [consultado 2019 03 29]Hong M.-E., Hwang S. K., Chang W. S., Kim B. W., Lee J., Sim S. J. Enhanced autotrophic astaxanthin production from Haematococcus pluvialis under high temperature via heat stress-driven Haber–Weiss reaction. Applied Microbiology and Biotechnology. [internet] 2015;99(12):5203–5215 [consultado 2019 06 09]Wan M, Zhang J, Hou D, Fan J, Li Y, Huang J, Wang J. The effect of temperature on cell growth and astaxanthin accumulation of Haematococcus pluvialis during a light–dark cyclic cultivation. Bioresour Technol [internet] 2014;167:276–283 [consultado 2019 06 09]Nguyen K. Astaxanthin: a comparative case of synthetic VS Natural production. Chemical and Biomolecular Engineering. [internet] 2013. [consultado 2019 04 01]Capelli B, Bagchi D, Cysewski G . Synthetic astaxanthin is significantly inferior to algal-based astaxanthin as an antioxidant and may not be suitable as a human nutraceutical supplement. Nutrafoods. [internet] 2013;12(4):145-152. [consultado 2019 04 01]Bai J, Beena B, Shashirekha V. Nomenclature,Taxonomy, Reproduction and Life Cycle of the genus Haematococcus, Haematococcaceae, Chlorophycea. Phykos. [internet] 2016;46(1):64-70. [consultado 2019 04 02]Peled E, Pick U, Zarka A, Shimoni E, Leu S, Boussiba S. Light-induced oil globule migration in haematococcus pluvialis (chlorophyceae). J Phycol. [internet] 2012;48(5):1209-1219. [consultado 2019 04 02]Lemoine Y, Schoefs B. Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress. Photosynth Res. [internet] 2010 Nov;106(1- 2):155-77. [consultado 2019 04 02]Shen Y., Cai M. G., Huang S. Y., et al. Haematococcus pluvialis culture in photobioreactor. Marine Sciences. [internet] 2010;34(10):83–89 . [consultado 2019 06 09]Sun H., Liu B., Lu X., Cheng K.-W., Chen F. Staged cultivation enhances biomass accumulation in the green growth phase of Haematococcus pluvialis. Bioresource Technology. [internet] 2017;233:326–331. [consultado 2019 06 09]Brinda BR, Sarada R, Kamath BS, Ravishankar GA. Accumulation of astaxanthin in flagellated cells of Haematococcus pluvialis – cultural and regulatory aspects. Curr Sci. [internet] 2004;87:1290–1295 [consultado 2019 04 03]Wang J., Han D., Sommerfeld M. R., Lu C., Hu Q. Effect of initial biomass density on growth and astaxanthin production of Haematococcus pluvialis in an outdoor photobioreactor. Journal of Applied Phycology. [internet] 2013;25(1):253–260 [consultado 2019 06 09]Li Y., Sommerfeld M., Chen F., Hu Q. Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae) Journal of Applied Phycology. [internet] 2010;22(3):253–263 [consultado 2019 06 09]Luo Q, Bian C, Tao M, Huang Y, Lv Y. Genome and Transcriptome Sequencing of the Astaxanthin-Producing Green Microalga, Haematococcus pluvialis. Genome Biol Evol.. [internet] 2019;11(1):166-173 [consultado 2019 06 09]Tufan M, Sayin S. The effects of iron and light intensity on biomass and pigment synthesis of heamotococcus pluvialis under laboratory conditions. ICAMS. [internet] 2014 [consultado 2019 04 03]Kobayashi M, Kakizono T, Nishio N, Nagai S. Effects of light intensity, light quality, and illumination cycle on astaxanthin formation in a green alga, Haematococcus pluvialis. Journal of Fermentation and Bioengineering. [internet] 1992;74(1):61-63 [consultado 2019 04 03]Kobayashi M, Kakizono T, Nagai S. Enhanced Carotenoid Biosynthesis by Oxidative Stress in Acetate-Induced Cyst Cells of a Green Unicellular Alga, Haematococcus pluvialis. Appl Environ Microbiol. [internet] 1993;59(3):867-73. [consultado 2019 04 03]Kobayashi M, Kurimura Y, Kakizono T, Nishio N, Tsuji Y. Morphological changes in the life cycle of the green alga Haematococcus pluvialis. Journal of Fermentation and Bioengineering. [internet] 1997;84(1):94-97 [consultado 2019 04 03]Li Y, Huang J, Sandmann G, Chen F. High‐light and sodium chloride stress differentially regulate the biosynthesis of astaxanthin in Chlorella zofingiensis (Chlorophyceae). Journal of Phycology. [internet] 2009;45(3) [consultado 2019 04 03]Meng CX, Teng CY, Jiang P, Qin S, Tseng C. Cloning and characterization of β- carotene ketolase gene promoter in Haematococcus pluvialis. Acta Biochim Biophys Sinica [internet] 2005; 37:270–275 [consultado 2019 04 03]Mittler R. Oxidative stress, antioxidants and stress tolerance. Trends in plant science. [internet] 2002;7(9):405-410 [consultado 2019 04 03]Olaizola M, Huntley ME. Recent advances in commercial production of astaxanthin from microalgae. Recent advances in marine biotechnology. [internet] 2003;9:143–164Orosa M, Valero J, Herrero C, Abalde J. Comparison of the accumulation of astaxanthin in Haematococcus pluvialis and other green microalgae under N- starvation and high light conditions. Biotechnology Letters. [internet] 2001;23(13):1079–1085 [consultado 2019 04 03]Park EK, Lee CG. Astaxanthin production by Haematococcus pluvialis under various light intensities and wavelengths. J Microbiol Biotechnol. [internet] 2001; 11:1024–1030 [consultado 2019 04 03]Pelah D, Sintov A, Cohen E. The effect of salt stress on the production of canthaxanthin and astaxanthin by Chlorella zofingiensis grown under limited light intensity. World J Microbiol Biotechnol. [internet] 2004; 20:483–486 [consultado 2019 04 03]Qiu B, Li Y. Photosynthetic acclimation and photoprotective mechanism in Haematococcus pluvialis (Chlorophyceae) during the accumulation of secondary carotenoids at elevated irradiation. Phycologia. [internet] 2006; 45:117–126 [consultado 2019 04 03]Khan Academy [internet] Estados Unidos; c2006. [consultado 2019 04 03]. Disponible en: https://es.khanacademy.org/science/biology/photosynthesis-in- plants/the-light-dependent-reactions-of-photosynthesis/a/light-dependent- reactionsSaini RK, Keum Y. Progress in microbial carotenoids production. Indian J Microbiol [internet] 2017;57:129–130 [consultado 2019 06 09]Mascia F, Girolomoni L, Alcocer M, Bargigia I, Perozeni F, Cazzaniga S, Cerullo G. Functional analysis of photosynthetic pigment binding complexes in the green alga Haematococcus pluvialis reveals distribution of astaxanthin in Photosystems. Sci Rep. [internet] 2017; 7: 16319. [consultado 2019 04 03]Bhosale P, Bernstein P. Microbial xanthophylls. Appl Microbiol Biotechnol. . [internet] 2015;68:445–455 [consultado 2019 06 09]Steinbrenner J, Linden H. Regulation of two carotenoid biosynthetic genes coding for phytoene synthase and carotenoid hydroxylase during stress-induced astaxanthin formation in the green alga Haematococcus pluvialis. Plant Physiol. [internet] 2001; 125:810–817. [consultado 2019 04 03]Steinbrenner J, Linden H (2003) Light induction of carotenoid biosynthesis genes in the green alga Haematococcus pluvialis: regulation by photosynthetic redox control. Plant Mol Biol. [internet] 2003; 52:343–356. [consultado 2019 04 03]Tan S, Cunningham FX, Youmans M, Grabowski B, Sun Z, Gantt E. Cytochrome f loss in astaxanthin-accumulating red cells of Haematococcus pluvialis (Chlorophyceae): comparison of photosynthetic activity, photosynthetic enzymes, and thylakoid membrane polypeptides in red and green cells. J Phycol. [internet] 1995; 31:897–905. [consultado 2019 04 03]Tran N-P, Park J-K, Lee C-G. Proteomic analysis of proteins in green alga Haematococcus pluvialis (Chlorophyceae) expressed under combined stress of nitrogen starvation and high irradiance. Enzyme Microb Technol. [internet] 2009; 45:241–246 [consultado 2019 04 03]Vidhyavathi R, Sarada R, Rhavishankar GA. Expression of carotenogenic genes and carotenoid production in Haematococcus pluvialis under the influence of carotenoid and fatty acid synthesis inhibitors. Enzyme Microb Technol. [internet] 2009; 45:88–93 [consultado 2019 04 03]Wang Y, Peng J. Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta). World J Microbiol Biotechnol. [internet] 2008; 24:1915–1922 [consultado 2019 04 03]Wang B, Zarka A, Trebst A, Boussiba S. Astaxanthin accumulation in Haematococcus pluvialis (Chlorophyceae) as an active photoprotective process during high irradiance. J Phycol. [internet] 2008; 39:1116–1124 [consultado 2019 04 03]factores de estrésestrés salinoluzmicroalgacarotenoidesORIGINALPRESENTACION.pdfPRESENTACION.pdfapplication/pdf1548953https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/1/PRESENTACION.pdf2ed531e689c6cd97c3c14ee272c91d7aMD51open accessIDENTIFICACION TRABAJO DE GRADO.pdfIDENTIFICACION TRABAJO DE GRADO.pdfapplication/pdf62511https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/2/IDENTIFICACION%20TRABAJO%20DE%20GRADO.pdf41ceb11ab802d4837c1d58fb598b747fMD52metadata only accessRODRIGUEZ ROMERO LAURA JOHANNA-.pdfRODRIGUEZ ROMERO LAURA JOHANNA-.pdfapplication/pdf1621353https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/3/RODRIGUEZ%20ROMERO%20LAURA%20JOHANNA-.pdf562b3ac7b65f0c69c5145adaf8179408MD53open accessLICENSElicense.txtlicense.txttext/plain; charset=utf-814828https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/4/license.txt2f9959eaf5b71fae44bbf9ec84150c7aMD54open accessTEXTPRESENTACION.pdf.txtPRESENTACION.pdf.txtExtracted texttext/plain10911https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/5/PRESENTACION.pdf.txtbbb6f7c592cdad8d1ba91c6ba028b87dMD55open accessIDENTIFICACION TRABAJO DE GRADO.pdf.txtIDENTIFICACION TRABAJO DE GRADO.pdf.txtExtracted texttext/plain5https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/7/IDENTIFICACION%20TRABAJO%20DE%20GRADO.pdf.txt14857a64d494d499c8ea946e1585dd67MD57metadata only accessRODRIGUEZ ROMERO LAURA JOHANNA-.pdf.txtRODRIGUEZ ROMERO LAURA JOHANNA-.pdf.txtExtracted texttext/plain200338https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/9/RODRIGUEZ%20ROMERO%20LAURA%20JOHANNA-.pdf.txte928b6577dd1ebbc27e785b9062b628aMD59open accessTHUMBNAILPRESENTACION.pdf.jpgPRESENTACION.pdf.jpgGenerated Thumbnailimage/jpeg7386https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/6/PRESENTACION.pdf.jpgc7173fd47663ed735410e8433392e006MD56open accessIDENTIFICACION TRABAJO DE GRADO.pdf.jpgIDENTIFICACION TRABAJO DE GRADO.pdf.jpgGenerated Thumbnailimage/jpeg6210https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/8/IDENTIFICACION%20TRABAJO%20DE%20GRADO.pdf.jpg7390c348849db7558b291e1adef7e63eMD58metadata only accessRODRIGUEZ ROMERO LAURA JOHANNA-.pdf.jpgRODRIGUEZ ROMERO LAURA JOHANNA-.pdf.jpgGenerated Thumbnailimage/jpeg6584https://repositorio.unicolmayor.edu.co/bitstream/unicolmayor/3709/10/RODRIGUEZ%20ROMERO%20LAURA%20JOHANNA-.pdf.jpgd2c143b1502cc5812ca08afc0a7de007MD510open accessunicolmayor/3709oai:repositorio.unicolmayor.edu.co:unicolmayor/37092021-11-24 03:03:25.557An error occurred on the license name.|||https://creativecommons.org/licenses/by-nc-sa/4.0/open accessBiblioteca Digital Unicolmayorrepositorio@unicolmayor.edu.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 |