Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization
Phycobiliproteins (PBPs) are a group of brilliant pigment proteins found in cyanobacteria and red algae; their synthesis and accumulation depend on several factors such as the type of strain employed, nutrient concentration, light intensity, light regimes, and others. This study evaluates the effect...
- Autores:
-
ZUORRO, Antonio
Leal Jerez, Angela Gisselle
Morales, Leidy
Mogollón Londoño, Sandra Oriana
Sánchez, Edwar
García-Martinez, Janet
Barajas Solano, andres F
- Tipo de recurso:
- Article of journal
- Fecha de publicación:
- 2021
- Institución:
- Universidad Francisco de Paula Santander
- Repositorio:
- Repositorio Digital UFPS
- Idioma:
- eng
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- oai:repositorio.ufps.edu.co:ufps/327
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- http://repositorio.ufps.edu.co/handle/ufps/327
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- openAccess
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- 2021 The Authors. Published by American Chemical Society
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Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| title |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| spellingShingle |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| title_short |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| title_full |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| title_fullStr |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| title_full_unstemmed |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| title_sort |
Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media Optimization |
| dc.creator.fl_str_mv |
ZUORRO, Antonio Leal Jerez, Angela Gisselle Morales, Leidy Mogollón Londoño, Sandra Oriana Sánchez, Edwar García-Martinez, Janet Barajas Solano, andres F |
| dc.contributor.author.none.fl_str_mv |
ZUORRO, Antonio Leal Jerez, Angela Gisselle Morales, Leidy Mogollón Londoño, Sandra Oriana Sánchez, Edwar García-Martinez, Janet Barajas Solano, andres F |
| description |
Phycobiliproteins (PBPs) are a group of brilliant pigment proteins found in cyanobacteria and red algae; their synthesis and accumulation depend on several factors such as the type of strain employed, nutrient concentration, light intensity, light regimes, and others. This study evaluates the effect of macronutrients (citrate buffer, NaNO3, K2HPO4, MgSO4, CaCl2, Na2CO3, and EDTA) and the concentration of trace metals in BG11 media on the accumulation of PBPs in a thermotolerant strain of Oscillatoria sp. The strain was grown in BG-11 media at 28 °C with a light:dark cycle of 12:12 h at 100 μmol m−2 s −1 for 15 days, and the effect of nutrients was evaluated using a Plackett−Burman Design followed by optimization using a response surface methodology. Results from the concentration of trace metals show that it can be reduced up to half-strength in its initial concentration without affecting both biomass and PBPs. Results from the Plackett−Burman Design revealed that only NaNO3, Na2CO3, and K2HPO4 show a significant increase in PBP production. Optimization employed a central Non-Factorial Response Surface Design with three levels and four factors (34 ) using NaNO3, Na2CO3, K2HPO4, and trace metals as variables, while the other components of BG-11 media (citrate buffer, MgSO4, CaCl2, and EDTA) were used in half of their initial concentration. Results from the optimization show that interaction between Na2CO3 and K2HPO4 highly increased PBPs’ concentration, with values of 15.21, 3.95, and 1.89 (% w/w), respectively. These results demonstrate that identifying and adjusting the concentration of critical nutrients can increase the concentration of PBPs up to two times for phycocyanin and allophycocyanin while four times for phycoerythrin. Finally, the reduction in non-key nutrients’ concentration will reduce the production costs of colorants at an industrial scale and increase the sustainability of the process. |
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2021 |
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2021-10-16T00:43:38Z |
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2021-10-16T00:43:38Z |
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2021-04-12 |
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ACS Omega ISSN: 2470-1343, 2021 vol:6 fasc: 16 págs: 10527 - 10536, DOI:10.1021/acsomega.0c04665 |
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Vol. 6, No. 16 (2021) |
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10536 |
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16 (2021) |
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Antonio Zuorro, Angela G. Leal-Jerez, Leidy K. Morales-Rivas, Sandra O. Mogollón-Londoño, Edwar M. Sanchez-Galvis, Janet B. García-Martínez, and Andrés F. Barajas-Solano ACS Omega 2021 6 (16), 10527-10536 DOI: 10.1021/acsomega.0c04665 |
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ACS Omega |
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2021 The Authors. Published by American Chemical Society |
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ZUORRO, Antonio1d9ebb9bdc0d09156364b9fd1169276a600Leal Jerez, Angela Gisselle3e88e414ba4874604c3c680c598ddcf6600Morales, Leidy009ee1ac04912cf69cba363938fdb49eMogollón Londoño, Sandra Oriana82aad7f548247cfb7c42fdf9c5a33d48600Sánchez, Edwar9f8f8dde369ab24867f08f331ecd62e6García-Martinez, Janet80edb67ba4cf51c8ef0d2bf62e0faa4d600Barajas Solano, andres F7237a5ca918751f9d045f15b62fd2f1f6002021-10-16T00:43:38Z2021-10-16T00:43:38Z2021-04-12http://repositorio.ufps.edu.co/handle/ufps/32710.1021/acsomega.0c04665Phycobiliproteins (PBPs) are a group of brilliant pigment proteins found in cyanobacteria and red algae; their synthesis and accumulation depend on several factors such as the type of strain employed, nutrient concentration, light intensity, light regimes, and others. This study evaluates the effect of macronutrients (citrate buffer, NaNO3, K2HPO4, MgSO4, CaCl2, Na2CO3, and EDTA) and the concentration of trace metals in BG11 media on the accumulation of PBPs in a thermotolerant strain of Oscillatoria sp. The strain was grown in BG-11 media at 28 °C with a light:dark cycle of 12:12 h at 100 μmol m−2 s −1 for 15 days, and the effect of nutrients was evaluated using a Plackett−Burman Design followed by optimization using a response surface methodology. Results from the concentration of trace metals show that it can be reduced up to half-strength in its initial concentration without affecting both biomass and PBPs. Results from the Plackett−Burman Design revealed that only NaNO3, Na2CO3, and K2HPO4 show a significant increase in PBP production. Optimization employed a central Non-Factorial Response Surface Design with three levels and four factors (34 ) using NaNO3, Na2CO3, K2HPO4, and trace metals as variables, while the other components of BG-11 media (citrate buffer, MgSO4, CaCl2, and EDTA) were used in half of their initial concentration. Results from the optimization show that interaction between Na2CO3 and K2HPO4 highly increased PBPs’ concentration, with values of 15.21, 3.95, and 1.89 (% w/w), respectively. These results demonstrate that identifying and adjusting the concentration of critical nutrients can increase the concentration of PBPs up to two times for phycocyanin and allophycocyanin while four times for phycoerythrin. Finally, the reduction in non-key nutrients’ concentration will reduce the production costs of colorants at an industrial scale and increase the sustainability of the process.10 páginasapplication/pdfengACS OmegaACS Omega ISSN: 2470-1343, 2021 vol:6 fasc: 16 págs: 10527 - 10536, DOI:10.1021/acsomega.0c04665Vol. 6, No. 16 (2021)1053616 (2021)105276Antonio Zuorro, Angela G. Leal-Jerez, Leidy K. Morales-Rivas, Sandra O. Mogollón-Londoño, Edwar M. Sanchez-Galvis, Janet B. García-Martínez, and Andrés F. Barajas-Solano ACS Omega 2021 6 (16), 10527-10536 DOI: 10.1021/acsomega.0c04665ACS Omega2021 The Authors. Published by American Chemical Societyhttps://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2https://pubs.acs.org/doi/10.1021/acsomega.0c04665Enhancement of Phycobiliprotein Accumulation in Thermotolerant Oscillatoria sp. through Media OptimizationArtículo de revistahttp://purl.org/coar/resource_type/c_6501http://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a85Kannaujiya, V. K.; Kumar, D.; Pathak, R. J.; Sonker, A. S.; Rajneesh.; Singh, V.; Sundaram, S., Sinha, R. P. Recent advances in production and the biotechnological significance of phycobiliproteins. In New Approaches in Biological Research; Sinha, R.P., Richa., Eds.; Nova Science Publisher: New York, 2017, pp. 1– 34.Google ScholarSantiago-Santos, M. 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 incorporada en las Obras Colectivas.

b.	Distribuir copias o fonogramas de las Obras, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública, incluyéndolas como incorporadas en Obras Colectivas, según corresponda.

c.	Distribuir copias de las Obras Derivadas que se generen, exhibirlas públicamente, ejecutarlas públicamente y/o ponerlas a disposición pública.
Los derechos mencionados anteriormente pueden ser ejercidos en todos los medios y formatos, actualmente conocidos o que se inventen en el futuro. Los derechos antes mencionados incluyen el derecho a realizar dichas modificaciones en la medida que sean técnicamente necesarias para ejercer los derechos en otro medio o formatos, pero de otra manera usted no está autorizado para realizar obras derivadas. Todos los derechos no otorgados expresamente por el Licenciante quedan por este medio reservados, incluyendo pero sin limitarse a aquellos que se mencionan en las secciones 4(d) y 4(e).

4. Restricciones.
La licencia otorgada en la anterior Sección 3 está expresamente sujeta y limitada por las siguientes restricciones:

a.	Usted puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra sólo bajo las condiciones de esta Licencia, y Usted debe incluir una copia de esta licencia o del Identificador Universal de Recursos de la misma con cada copia de la Obra que distribuya, exhiba públicamente, ejecute públicamente o ponga a disposición pública. No es posible ofrecer o imponer ninguna condición sobre la Obra que altere o limite las condiciones de esta Licencia o el ejercicio de los derechos de los destinatarios otorgados en este documento. No es posible sublicenciar la Obra. Usted debe mantener intactos todos los avisos que hagan referencia a esta Licencia y a la cláusula de limitación de garantías. Usted no puede distribuir, exhibir públicamente, ejecutar públicamente, o poner a disposición pública la Obra con alguna medida tecnológica que controle el acceso o la utilización de ella de una forma que sea inconsistente con las condiciones de esta Licencia. Lo anterior se aplica a la Obra incorporada a una Obra Colectiva, pero esto no exige que la Obra Colectiva aparte de la obra misma quede sujeta a las condiciones de esta Licencia. Si Usted crea una Obra Colectiva, previo aviso de cualquier Licenciante debe, en la medida de lo posible, eliminar de la Obra Colectiva cualquier referencia a dicho Licenciante o al Autor Original, según lo solicitado por el Licenciante y conforme lo exige la cláusula 4(c).

b.	Usted no puede ejercer ninguno de los derechos que le han sido otorgados en la Sección 3 precedente de modo que estén principalmente destinados o directamente dirigidos a conseguir un provecho comercial o una compensación monetaria privada. El intercambio de la Obra por otras obras protegidas por derechos de autor, ya sea a través de un sistema para compartir archivos digitales (digital file-sharing) o de cualquier otra manera no será considerado como estar destinado principalmente o dirigido directamente a conseguir un provecho comercial o una compensación monetaria privada, siempre que no se realice un pago mediante una compensación monetaria en relación con el intercambio de obras protegidas por el derecho de autor.

c.	Si usted distribuye, exhibe públicamente, ejecuta públicamente o ejecuta públicamente en forma digital la Obra o cualquier Obra Derivada u Obra Colectiva, Usted debe mantener intacta toda la información de derecho de autor de la Obra y proporcionar, de forma razonable según el medio o manera que Usted esté utilizando: (i) el nombre del Autor Original si está provisto (o seudónimo, si fuere aplicable), y/o (ii) el nombre de la parte o las partes que el Autor Original y/o el Licenciante hubieren designado para la atribución (v.g., un instituto patrocinador, editorial, publicación) en la información de los derechos de autor del Licenciante, términos de servicios o de otras formas razonables; el título de la Obra si está provisto; en la medida de lo razonablemente factible y, si está provisto, el Identificador Uniforme de Recursos (Uniform Resource Identifier) que el Licenciante especifica para ser asociado con la Obra, salvo que tal URI no se refiera a la nota sobre los derechos de autor o a la información sobre el licenciamiento de la Obra; y en el caso de una Obra Derivada, atribuir el crédito identificando el uso de la Obra en la Obra Derivada (v.g., "Traducción Francesa de la Obra del Autor Original," o "Guión Cinematográfico basado en la Obra original del Autor Original"). Tal crédito puede ser implementado de cualquier forma razonable; en el caso, sin embargo, de Obras Derivadas u Obras Colectivas, tal crédito aparecerá, como mínimo, donde aparece el crédito de cualquier otro autor comparable y de una manera, al menos, tan destacada como el crédito de otro autor comparable.

d.	Para evitar toda confusión, el Licenciante aclara que, cuando la obra es una composición musical:

i.	Regalías por interpretación y ejecución bajo licencias generales. El Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública o la ejecución pública digital de la obra y de recolectar, sea individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, SAYCO), las regalías por la ejecución pública o por la ejecución pública digital de la obra (por ejemplo Webcast) licenciada bajo licencias generales, si la interpretación o ejecución de la obra está primordialmente orientada por o dirigida a la obtención de una ventaja comercial o una compensación monetaria privada.

ii.	Regalías por Fonogramas. El Licenciante se reserva el derecho exclusivo de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, los consagrados por la SAYCO), una agencia de derechos musicales o algún agente designado, las regalías por cualquier fonograma que Usted cree a partir de la obra (“versión cover”) y distribuya, en los términos del régimen de derechos de autor, si la creación o distribución de esa versión cover está primordialmente destinada o dirigida a obtener una ventaja comercial o una compensación monetaria privada.

e.	Gestión de Derechos de Autor sobre Interpretaciones y Ejecuciones Digitales (WebCasting). Para evitar toda confusión, el Licenciante aclara que, cuando la obra sea un fonograma, el Licenciante se reserva el derecho exclusivo de autorizar la ejecución pública digital de la obra (por ejemplo, webcast) y de recolectar, individualmente o a través de una sociedad de gestión colectiva de derechos de autor y derechos conexos (por ejemplo, ACINPRO), las regalías por la ejecución pública digital de la obra (por ejemplo, webcast), sujeta a las disposiciones aplicables del régimen de Derecho de Autor, si esta ejecución pública digital está primordialmente dirigida a obtener una ventaja comercial o una compensación monetaria privada.

5. Representaciones, Garantías y Limitaciones de Responsabilidad.
A MENOS QUE LAS PARTES LO ACORDARAN DE OTRA FORMA POR ESCRITO, EL LICENCIANTE OFRECE LA OBRA (EN EL ESTADO EN EL QUE SE ENCUENTRA) “TAL CUAL”, SIN BRINDAR GARANTÍAS DE CLASE ALGUNA RESPECTO DE LA OBRA, YA SEA EXPRESA, IMPLÍCITA, LEGAL O CUALQUIERA OTRA, INCLUYENDO, SIN LIMITARSE A ELLAS, GARANTÍAS DE TITULARIDAD, COMERCIABILIDAD, ADAPTABILIDAD O ADECUACIÓN A PROPÓSITO DETERMINADO, AUSENCIA DE INFRACCIÓN, DE AUSENCIA DE DEFECTOS LATENTES O DE OTRO TIPO, O LA PRESENCIA O AUSENCIA DE ERRORES, SEAN O NO DESCUBRIBLES (PUEDAN O NO SER ESTOS DESCUBIERTOS). ALGUNAS JURISDICCIONES NO PERMITEN LA EXCLUSIÓN DE GARANTÍAS IMPLÍCITAS, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.

6. Limitación de responsabilidad.
A MENOS QUE LO EXIJA EXPRESAMENTE LA LEY APLICABLE, EL LICENCIANTE NO SERÁ RESPONSABLE ANTE USTED POR DAÑO ALGUNO, SEA POR RESPONSABILIDAD EXTRACONTRACTUAL, PRECONTRACTUAL O CONTRACTUAL, OBJETIVA O SUBJETIVA, SE TRATE DE DAÑOS MORALES O PATRIMONIALES, DIRECTOS O INDIRECTOS, PREVISTOS O IMPREVISTOS PRODUCIDOS POR EL USO DE ESTA LICENCIA O DE LA OBRA, AUN CUANDO EL LICENCIANTE HAYA SIDO ADVERTIDO DE LA POSIBILIDAD DE DICHOS DAÑOS. ALGUNAS LEYES NO PERMITEN LA EXCLUSIÓN DE CIERTA RESPONSABILIDAD, EN CUYO CASO ESTA EXCLUSIÓN PUEDE NO APLICARSE A USTED.

7. Término.

a.	Esta Licencia y los derechos otorgados en virtud de ella terminarán automáticamente si Usted infringe alguna condición establecida en ella. Sin embargo, los individuos o entidades que han recibido Obras Derivadas o Colectivas de Usted de conformidad con esta Licencia, no verán terminadas sus licencias, siempre que estos individuos o entidades sigan cumpliendo íntegramente las condiciones de estas licencias. Las Secciones 1, 2, 5, 6, 7, y 8 subsistirán a cualquier terminación de esta Licencia.

b.	Sujeta a las condiciones y términos anteriores, la licencia otorgada aquí es perpetua (durante el período de vigencia de los derechos de autor de la obra). No obstante lo anterior, el Licenciante se reserva el derecho a publicar y/o estrenar la Obra bajo condiciones de licencia diferentes o a dejar de distribuirla en los términos de esta Licencia en cualquier momento; en el entendido, sin embargo, que esa elección no servirá para revocar esta licencia o que deba ser otorgada , bajo los términos de esta licencia), y esta licencia continuará en pleno vigor y efecto a menos que sea terminada como se expresa atrás. La Licencia revocada continuará siendo plenamente vigente y efectiva si no se le da término en las condiciones indicadas anteriormente.

8. Varios.

a.	Cada vez que Usted distribuya o ponga a disposición pública la Obra o una Obra Colectiva, el Licenciante ofrecerá al destinatario una licencia en los mismos términos y condiciones que la licencia otorgada a Usted bajo esta Licencia.

b.	Si alguna disposición de esta Licencia resulta invalidada o no exigible, según la legislación vigente, esto no afectará ni la validez ni la aplicabilidad del resto de condiciones de esta Licencia y, sin acción adicional por parte de los sujetos de este acuerdo, aquélla se entenderá reformada lo mínimo necesario para hacer que dicha disposición sea válida y exigible.

c.	Ningún término o disposición de esta Licencia se estimará renunciada y ninguna violación de ella será consentida a menos que esa renuncia o consentimiento sea otorgado por escrito y firmado por la parte que renuncie o consienta.

d.	Esta Licencia refleja el acuerdo pleno entre las partes respecto a la Obra aquí licenciada. No hay arreglos, acuerdos o declaraciones respecto a la Obra que no estén especificados en este documento. El Licenciante no se verá limitado por ninguna disposición adicional que pueda surgir en alguna comunicación emanada de Usted. Esta Licencia no puede ser modificada sin el consentimiento mutuo por escrito del Licenciante y Usted.
0000-0002-8173-38091d9ebb9bdc0d09156364b9fd1169276a6000000-0001-6719-740880edb67ba4cf51c8ef0d2bf62e0faa4d6000000-0003-2765-91317237a5ca918751f9d045f15b62fd2f1f600 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