WO2022124878A1 - Method for obtaining carotene-free chlorella vulgaris microalgae - Google Patents
Method for obtaining carotene-free chlorella vulgaris microalgae Download PDFInfo
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- WO2022124878A1 WO2022124878A1 PCT/MX2021/050084 MX2021050084W WO2022124878A1 WO 2022124878 A1 WO2022124878 A1 WO 2022124878A1 MX 2021050084 W MX2021050084 W MX 2021050084W WO 2022124878 A1 WO2022124878 A1 WO 2022124878A1
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- microalgae
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- chlorella vulgaris
- photobioreactor
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Abstract
Disclosed is a method for obtaining carotene-free Chlorella vulgaris microalgae. The method comprises: adding the microalga Chlorella vulgaris, in the exponential growth phase, to an "f" culture medium; placing the microalgal culture in a tubular photobioreactor arranged in the centre of a compound parabolic concentrator; maintaining the culture at a maximum temperature of between 33 and 35 °C and a maximum irradiance of 5090 µmol m2 s- 1; centrifuging the biomass; and lyophilising the centrifuged biomass.
Description
MÉTODO PARA OBTENER MICROALGAS CHLORELLA VULGARIS LIBRES DE CAROTENOS METHOD TO OBTAIN CAROTEN FREE CHLORELLA VULGARIS MICROALGAE
Campo de la invención field of invention
La presente invención se refiere a un método para incrementar la concentración de lipidos en microalgas y más particularmente se refiere a un método controlado para incrementar la concentración de lipidos en células de la microalga Chl orella vulgari s que utili za radiación solar recolectada a través de un sistema concentrador de energía integrado a un sistema de f otobiorreactor tubular, que permite obtener células con baj o contenido de pigmentos y favorece la producción de lipidos precursores de biodiesel , de manera que al ser usadas en la producción de biodiesel no hay pigmentos residuales en el biocombustible , por lo que produzcan menos residuos en los motores . The present invention refers to a method to increase the concentration of lipids in microalgae and more particularly refers to a controlled method to increase the concentration of lipids in cells of the microalga Chl orella vulgaris that uses solar radiation collected through a energy concentrator system integrated into a tubular photobioreactor system, which allows cells with a low content of pigments to be obtained and favors the production of biodiesel precursor lipids, so that when they are used in the production of biodiesel there are no residual pigments in the biofuel , so they produce less waste in the engines .
Antecedentes de la invención Background of the invention
El cultivo de microalgas se reali za de forma autotrófica al suministrar aporte nutritivo y luz a las células en los cultivos para que se promueva el incremento en el número de células y la cantidad de biomasa . Los cultivos de microalgas en condiciones controladas , se reali zan al interior en edi ficaciones en laboratorios especiali zados y en sistemas en donde se provee luz arti ficial , nutrientes y dependiendo del volumen del cultivo se suministra aireación para mantener las células en suspensión en la columna de agua . Los cultivos de microalgas mantenidos al exterior , usualmente son sistemas en donde se utiliza la luz natural y sólo se suministran nutrientes y aireación . Para los cultivos de microalgas mantenidos al interior , es necesario suministrar iluminación arti ficial que usualmente es provista por medio de lámparas de distintos tipos ( fluorescentes , halógena, LEDs entre otras )
[Garibay-Hernández, A., R. Vázquez-Duhalt , M.P. Sánchez- Saavedra, Serrano-Carreón, L. y A. Martinez- Jiménez . (2009) . Biodiesel a partir de microalgas. BioTecnologia Revista de la Sociedad Mexicana de Biotecnología y Bioingenierí a . 13 (3) : 38- 61.] . Para los cultivos de microalgas mantenidos al exterior, la iluminación es provista por el sol, por lo que el cultivo está expuesto a condiciones cambiantes de irradiancia que varían según la época del año. Dependiendo de las condiciones de iluminación (irradiancia y composición espectral) , se pueden producir cambios en la tasa de crecimiento, producción de biomasa y en la composición proximal de las diversas especies de microalgas [Romero-Romero, C.C., Sánchez-Saavedra, M.P. (2017) . Effect of light quality on the growth and proximal composition of Amphora sp. Journal of Applied Phycology 29:1203-1211. doi : 10.1007/sl 0811-016-1029-7 ] . The cultivation of microalgae is carried out autotrophically by supplying nutrients and light to the cells in the cultures so that the increase in the number of cells and the amount of biomass is promoted. Microalgae cultures under controlled conditions are carried out indoors in buildings in specialized laboratories and in systems where artificial light and nutrients are provided and, depending on the volume of the culture, aeration is provided to keep the cells in suspension in the column. of water . Outdoor microalgae cultures are usually systems where natural light is used and only nutrients and aeration are provided. For microalgae cultures kept indoors, it is necessary to provide artificial lighting that is usually provided by means of different types of lamps (fluorescent, halogen, LEDs, among others). [Garibay-Hernández, A., R. Vázquez-Duhalt, MP Sánchez-Saavedra, Serrano-Carreón, L. and A. Martinez-Jiménez. (2009). Biodiesel from microalgae. BioTecnologia Journal of the Mexican Society of Biotechnology and Bioengineering. 13 (3) : 38-61.] . For microalgae cultures kept outdoors, the lighting is provided by the sun, so the culture is exposed to changing conditions of irradiance that vary according to the time of year. Depending on the lighting conditions (irradiance and spectral composition), changes can occur in the growth rate, biomass production and in the proximal composition of the various species of microalgae [Romero-Romero, CC, Sánchez-Saavedra, MP ( 2017). Effect of light quality on the growth and proximal composition of Amphora sp. Journal of Applied Phycology 29:1203-1211. doi:10.1007/sl0811-016-1029-7].
La luz tiene un papel fundamental en el desarrollo de los cultivos de diversas especies de microalgas. La luz puede variar en intensidad ( f otoperíodo ) y composición espectral, y ambos factores modifican la fotosíntesis y las rutas metabólicas de cultivos de diversas especies de microalgas y causan cambios en la tasa de crecimiento, en la producción de biomasa y en la composición bioquímica [Muller-Feuga, A. , J Moal, R KaasRobert, R., Cahu, C., Robin, J., Divanach, P. (2003) . The microalgae of aquaculture. 206-252. Uses of En: Live feeds in marine aquaculture. Stottrup, J.G. y L.A. McEvoy (eds) . Blackwell Publishing, Oxford. 318 pp.1-3; Khajepour, F., Hosseini, S.A., Nasrabadi, R.G., Markou, G. (2015) Effect of light intensity and photoperiod on growth and biochemical composition of a local isolate of Nostoc caldcóla. Applied Biochemestry Biotechnolology 6 ( 8 ) : 2279-2289 ] . Light plays a fundamental role in the development of cultures of various species of microalgae. Light can vary in intensity (photoperiod) and spectral composition, both of which modify the photosynthesis and metabolic pathways of cultures of various microalgae species and cause changes in growth rate, biomass production, and biochemical composition. [Muller-Feuga, A., J Moal, R. KaasRobert, R., Cahu, C., Robin, J., Divanach, P. (2003). The microalgae of aquaculture. 206-252. Uses of En: Live feeds in marine aquaculture. Stottrup, J.G. and the. McEvoy (eds). Blackwell Publishing, Oxford. 318 pp.1-3; Khajepour, F., Hosseini, S.A., Nasrabadi, R.G., Markou, G. (2015) Effect of light intensity and photoperiod on growth and biochemical composition of a local isolate of Nostoc caldcóla. Applied Biochemistry Biotechnology 6(8):2279-2289].
La respuesta f otosintética de las microalgas a los cambios de la luz se puede describir por medio de la curva de Fotosintes (P) e irradiancia (I) o bien curvas P/I, y se ha utilizado como una herramienta para analizar la respuesta del
aparato f otosintético al estrés ambiental . Los parámetros f otosintéticos que describen la curva P/ I se utili zan para la descripción de la eficiencia del uso de fotones de luz por las células de las microalgas [Vonshak, A. , G, Torzillo . ( 2005 ) . Environmental stress physiology . 57 - 82 . Environmental stress physiology . En : Richomnd A ( ed) . Handbook of microalgal culture . Biotechnology and Applied Phycology . Blackwell Publishing, Oxford . 566 pp . 4 ] . La disponibilidad de luz en el cultivo de microalgas se modi fica a lo largo del tiempo de cultivo , debido a la relación inversa entre la disponibilidad de luz y la concentración celular . Por lo anterior, es importante proporcionar suficiente luz para favorecer el crecimiento de las células de las diversas especies de microalgas ; sin embargo , el aumento de la luz en condiciones de cultivo controladas es mediante la ampliación del número de lámparas utili zadas . El costo energético por el uso de energía para las lámparas , es el principal impedimento relacionado con la producción de las microalgas en condiciones de cultivo controladas . La importancia de conocer las curvas P/ I de las cepas de microalgas , proporciona información importante sobre las condiciones de iluminación requeridas tanto para los sistemas de cultivo mantenidos al interior como en los del exterior . The photosynthetic response of microalgae to changes in light can be described by means of the photosynthetic (P) and irradiance (I) curve or P/I curves, and has been used as a tool to analyze the response of the algae. photosynthetic apparatus to environmental stress. The photosynthetic parameters that describe the P/I curve are used for the description of the efficiency of the use of photons of light by the cells of microalgae [Vonshak, A. , G, Torzillo . (2005). Environmental stress physiology. 57 - 82 . Environmental stress physiology. In: Richmond A (ed). Handbook of microalgal culture. Biotechnology and Applied Phycology. Blackwell Publishing, Oxford. 566pp. 4 ] . The availability of light in the culture of microalgae is modified throughout the culture time, due to the inverse relationship between the availability of light and the cell concentration. Therefore, it is important to provide enough light to favor the growth of the cells of the various species of microalgae; however , the increase in light under controlled growing conditions is by expanding the number of lamps used . The energy cost for the use of energy for the lamps is the main impediment related to the production of microalgae in controlled culture conditions. The importance of knowing the P/I curves of microalgae strains provides important information on the lighting conditions required for both indoor and outdoor cultivation systems.
Existen diversos tipos de lámparas que se han utili zado para la iluminación de los cultivos de microalgas , entre otras se destacan las lámparas fluorescentes , lámparas halógenas y lámparas de diodo emisor de luz . Cada uno de los diversos tipos de lámparas usadas para el cultivo de las microalgas tiene ciertas ventaj as y desventaj as que van desde la generación de calor, distinta durabilidad, dieferencias en la magnitud de la irradiancia suministrada y cambios en la composición espectral de la luz . There are various types of lamps that have been used to illuminate microalgae cultures, among others, fluorescent lamps, halogen lamps and light-emitting diode lamps. Each of the various types of lamps used for the cultivation of microalgae has certain advantages and disadvantages ranging from heat generation, different durability, differences in the magnitude of irradiance supplied and changes in the spectral composition of the light. .
El cultivo de microalgas puede reali zarse en sistemas de f otobioreactor tubular, f otobioreactor plano o en sistemas de
estanques. Los f otobioreactores tubulares consisten de tubos transparentes de diferente diámetros y que pueden ser de materiales diversos como vidrio, acrilico y plexiglass entre otros materiales que permitan el paso de la luz. Los f otobioreactores planos están construidos con láminas y el ancho del sistema es menor que las caras de las láminas. El material con el cual se construyen los f otobioreactores planos es usualmente acrilico. Los estanques pueden ser de forma circular, rectangular y cuadrada. Los materiales con los cuales se construyen los estanques son acrilico, fibra de vidrio, plástico, cemento y vinil entre otros tipos de materiales que pueden ser translúcidos u opacos. El suministro de la iluminación en los sistemas tubulares y planos es instalado usualmente en el supperficie de mayor área del sistema de cultivo, en otros casos, la iluminación a las células de microalgas es provista al interior o desde el exterior del sistema. Mientras que en los sistemas de estanques, la iluminación es suministrada ya sea en la superficie del volumen de agua cuando se usan sistemas opacos, o al interior del sistema. En los estanques trasparentes la iluminación es suministrada usualmente en el área de mayor exposición . The cultivation of microalgae can be carried out in tubular photobioreactor systems, flat photobioreactor systems or in ponds. Tubular photobioreactors consist of transparent tubes of different diameters and can be made of various materials such as glass, acrylic and plexiglass, among other materials that allow the passage of light. Planar photobioreactors are built with plates and the width of the system is less than the faces of the plates. The material with which flat photobioreactors are built is usually acrylic. Ponds can be circular, rectangular and square. The materials with which the ponds are built are acrylic, fiberglass, plastic, cement and vinyl, among other types of materials that can be translucent or opaque. The lighting supply in tubular and flat systems is usually installed on the surface of the largest area of the culture system, in other cases, the lighting to the microalgae cells is provided inside or from the outside of the system. Whereas in pond systems, lighting is provided either on the surface of the water body when opaque systems are used, or within the system. In transparent ponds lighting is usually provided in the area of greatest exposure.
Se ha encontrado que diversas especies de microalgas al ser mantenidas en altos niveles de irradiancia, producen un incremento en la sintesis de lipidos y ácidos grasos precursosres para la producción de biodiésel, la magnitud de la irradiancia utilizada varia en valores que van desde los 400 hasta los 900 pmol
y depende las características fisiológicas de las especies de microalga [He, Q. , Yang, H., Wu, L., Hu, C. (2015) . Effect of light intensity on physiological changes, carbon allocation and neutral lipid accumulation in oleaginous microalgae. Bioresource Technology 191:219-228; Kim, D.W., Shin, W., Sung, M., Lee, B., Chang, Y.K. (2019) . Light intensity control as a strategy to improve
lipid productivity in Chlorella sp. HS2 for biodiesel production. Biomass and Bioenergy 126:211-219.] . Asimsimo, se ha investigado el efecto de la influencia de la temperatura en cultivos de Chlorella vulgaris mantenidos en un sistema de f otobioreactor al exterior con iluminación del sol, a fin de observar el efecto de las altas temperaturas que se pueden alcanzar en el verano. Se realizaron cultivos de Chlorella vulgaris en medio 3N-Bristol en un f otobioreactor cilindro- cónico de 2.4 litros en un sistema multicultivador (PSI MC- 1000 Photon systems Instruments) . La iluminación fue provista por lamparas fluorescentes blanca y rosa a 100 pmol
s , con adición de aire y 2% de CO2 en un flujo continuo de 220 ml min-1. Los ensayos se realizaron a 25°C y los ensayos de reanimación se realizaron a 35°C y 30°C por 3 dias y después la temperatura se mantuvo a 25°C. Se encontró que la viabilidad (Vi) de las células de Chlorella vulgaris disminuye con el incremento de los valores de temperatura utilizados: 20°C (Vi:98.7%) , 25°C (Vi:74.1%) , 28°C (Vi:41.8%) y 30°CIt has been found that various species of microalgae, when maintained at high levels of irradiance, produce an increase in the synthesis of lipids and fatty acids precursors for the production of biodiesel, the magnitude of the irradiance used varies in values ranging from 400 to the 900 pmol and it depends on the physiological characteristics of the microalgae species [He, Q. , Yang, H., Wu, L., Hu, C. (2015) . Effect of light intensity on physiological changes, carbon allocation and neutral lipid accumulation in oleaginous microalgae. Bioresource Technology 191:219-228; Kim DW, Shin W, Sung M, Lee B, Chang YK (2019). Light intensity control as a strategy to improve lipid productivity in Chlorella sp. HS2 for biodiesel production. Biomass and Bioenergy 126:211-219.] . Likewise, the effect of the influence of temperature on cultures of Chlorella vulgaris maintained in a photobioreactor system outdoors with sunlight has been investigated, in order to observe the effect of the high temperatures that can be reached in the summer. Chlorella vulgaris cultures were grown in 3N-Bristol medium in a 2.4 liter cylindrical-conical photobioreactor in a multicultivator system (PSI MC-1000 Photon systems Instruments). Illumination was provided by white and pink fluorescent lamps at 100 pmol. s, with addition of air and 2% CO2 in a continuous flow of 220 ml min -1 . The tests were carried out at 25°C and the resuscitation tests were carried out at 35°C and 30°C for 3 days and then the temperature was maintained at 25°C. It was found that the viability (Vi) of Chlorella vulgaris cells decreases with increasing temperature values used: 20°C (Vi:98.7%), 25°C (Vi:74.1%), 28°C (Vi :41.8%) and 30°C
(Vi: 20.1%) . Los resultados mostraron que la temperatura produce f otoaclimatación en la fracción viable de las células en los valores de altas temperaturas (28 °C) . Por lo anterior, las células tuvieron un decremento del contenido de clorofila a (Cío a) de 20°C a 25°C (Cío a:l%) y de 25°C a 28°C (Cío a: 75%) . Mientras que el contenido de la Clorofila b aumentó de 20°C a 25°C (Cío b:7%) y de 25°C a 28°C disminuyó (Cío b:71%) . La composición elemental de las células se modificó por efecto de la temperatura: el contenido de carbono (C) , nitrógeno (N) e hidrógeno tuvieron una relación inversa con respecto a la temperatura (20°C,25°C y 28°C) , mientras que el contenido azufre y la relación C/N tuvieron una relación directa respecto a las temperaturas utilizadas. De lo anterior se concluye que la productividad es fuertemente reducida cuando la temperatura es mayor que los niveles óptimos de temperatura y la eficiencia del proceso de conversión es
fuertemente afectado [Serra-Maia, R., Bernard, O., Gonqalves, A., Bensalem, S., Lopes, F. (2016) . Influence of temperature on Chlorella vulgaris growth and mortality rates in a photobioreactor. Algal Research 18:352-359] . (I saw: 20.1%). The results showed that the temperature produces photoacclimation in the viable fraction of the cells at high temperature values (28 °C). Therefore, the cells had a decrease in chlorophyll a content (Cío a) from 20°C to 25°C (Cío a: 1%) and from 25°C to 28°C (Cío a: 75%). While the content of Chlorophyll b increased from 20°C to 25°C (Cío b:7%) and from 25°C to 28°C it decreased (Cío b:71%). The elemental composition of the cells was modified by the effect of temperature: the content of carbon (C), nitrogen (N) and hydrogen had an inverse relationship with respect to temperature (20°C, 25°C and 28°C). , while the sulfur content and the C/N ratio had a direct relationship with the temperatures used. From the above it is concluded that the productivity is strongly reduced when the temperature is higher than the optimum temperature levels and the efficiency of the conversion process is strongly affected [Serra-Maia, R., Bernard, O., Gonqalves, A., Bensalem, S., Lopes, F. (2016). Influence of temperature on Chlorella vulgaris growth and mortality rates in a photobioreactor. Algal Research 18:352-359].
Una de las opciones que se pueden usar para aumentar la irradiancia sobre los cultivos de microalgas, es el uso de concentradores parabólicos compuestos (CPC) , los cuales han sido utilizados como sistemas concentradores de energía lumínica, que promueven el incremento de la temperatura del fluido que es irradiado. Sin embargo, las temperaturas que se producen por el uso de los CPC convencionalente van de los 60°C a 100°C, en dependencia de las características del sistema. El uso de los CPC ha sido evaluado para el tratamiento de aguas residuales, ya que las altas temperaturas que produce el sistema actúan como elemento de desinfección y eliminan bacterias [Cabrera, A., Miralles, S., Santos- Juanes , L. (2019) . Solar Water Detoxification. En: Solar Resources Mapping (pp. 341-351) . Springer, Cham.; Fendrich, M., Quaranta, A., Orlandi, M., Bettonte, M., Miotello, A. (2019) . Solar Concentration for Wastewaters Remediation: A Review of Materials and Technologies. Applied Sciences 9(1) :118. 26 pp . ] . Otro uso de los concentradores solares de energía es para el proceso de destilación de agua [Arunkumar, T., Velraj , R., Ahsan, A., Khalifa, A. J. N., Shams, S., Denkenberger , D., Sathyamurthy, R. (2016) . Effect of parabolic solar energy collectors for water distillation. Desalination and Water Treatment 57 ( 45 ) : 21234-21242 ] . Además, los sistemas de CPC han sido utilizados para la producción de hidrógeno [Cao, F., Wei, Q. , Liu, H., Lu, N., Zhao, L., Guo, L. (2018) . Development of the direct solar photocatalytic water splitting system for hydrogen production in Northwest China: Design and evaluation of photoreactor. Renewable Energy 121:153-163] . Sin embargo, el valor de la máxima temperatura para el mantenimiento de los cultivos de Chlorella vulgaris debe ser menor a 35°C, ya que a
valores mayores de temperatura las células mueren (Cassidy, 2011; Daliry et al. 2017) . [Cassidy, K. O. (2011) . Evaluating algal growth at different temperatures. Thesis and Dissertations-Biosystems and Agricultural Engineering. University of Kentucky. USA. 59 pp . ] [Daliry, S., Hallajisani, A., Mohammadi, R. J., Nouri, H., & Golzary, A. (2017) . Investigation of optimal condition for Chlorella vulgaris microalgae growth. Global Journal Environmental Science and Management 3 (2) :217-230] , por lo que el uso de los CPC para el cultivo de microalgas ha sido poco desarrollado debido a las limitantes que su uso conlleva. One of the options that can be used to increase the irradiance on microalgae cultures is the use of compound parabolic concentrators (CPC), which have been used as light energy concentrating systems, which promote an increase in fluid temperature. which is irradiated. However, the temperatures produced by the use of CPCs conventionally range from 60°C to 100°C, depending on the characteristics of the system. The use of CPCs has been evaluated for wastewater treatment, since the high temperatures produced by the system act as a disinfection element and eliminate bacteria [Cabrera, A., Miralles, S., Santos- Juanes , L. ( 2019). Solar Water Detoxification. In: Solar Resources Mapping (pp. 341-351). Springer, Cham.; Fendrich, M., Quaranta, A., Orlandi, M., Bettonte, M., Miotello, A. (2019). Solar Concentration for Wastewaters Remediation: A Review of Materials and Technologies. Applied Sciences 9(1):118. 26pp. ] . Another use of solar power concentrators is for the water distillation process [Arunkumar, T., Velraj, R., Ahsan, A., Khalifa, AJN, Shams, S., Denkenberger, D., Sathyamurthy, R. (2016). Effect of parabolic solar energy collectors for water distillation. Desalination and Water Treatment 57(45):21234-21242]. Furthermore, CPC systems have been used for hydrogen production [Cao, F., Wei, Q., Liu, H., Lu, N., Zhao, L., Guo, L. (2018). Development of the direct solar photocatalytic water splitting system for hydrogen production in Northwest China: Design and evaluation of photoreactor. Renewable Energy 121:153-163]. However, the value of the maximum temperature for the maintenance of Chlorella vulgaris cultures must be less than 35°C, since at higher temperature values, the cells die (Cassidy, 2011; Daliry et al. 2017). [Cassidy, K.O. (2011). Evaluating algal growth at different temperatures. Thesis and Dissertations-Biosystems and Agricultural Engineering. University of Kentucky. USES. 59pp. ] [Daliry, S., Hallajisani, A., Mohammadi, R.J., Nouri, H., & Golzary, A. (2017). Investigation of optimal condition for Chlorella vulgaris microalgae growth. Global Journal Environmental Science and Management 3 (2) :217-230] , so the use of CPCs for the cultivation of microalgae has been little developed due to the limitations that their use entails.
A fin de mejorar las condiciones de cultivo de las microalgas, se han desarrollado varias propuestas enfocadas a modificar las concentraciones de compuestos de interés en las microalgas, tales como la descrita en la solicitud de patente US20100255458A1, en la que se menciona el uso de concentradores parabólicos compuestos como estrategia para mejorar el nivel de irradiancia y mejorar el área efectiva de la luz que incide sobre los cultivos realizados en f otobiorreactor . En dicha solicitud se indica que los cultivos pueden ser de organismos f otoautótrof os , pero no describe un tipo de organismo en particular. Esta invención describe sistemas de manejo automatizado que permiten controlar las condiciones del cultivo. Asimismo, se describe que el sistema propuesto podría ser utilizado en un sistema con iluminación con lámparas o al exterior. Para controlar la temperatura del medio liquido, describe el uso de celdas de Peltier, pero no se indica el número de celdas o la potencia de estos elementos, tampoco se detalla la ubicación de las celdas en el sistema. Esta invención no indica el flujo de medio liquido, tampoco la ubicación del CPC y del f otobiorreactor . En el diseño propuesto en la solicitud arriba citada, no se indica el rango de las condiciones en las que puede trabajar el sistema y tampoco el tipo de organismos f otoautotróf icos que
podrían ser mantenidos en el sistema. La descripción de la invención es muy general y no muestra datos específicos del uso de este tipo de sistema y tampoco la calidad de la biomasa producida . In order to improve the cultivation conditions of microalgae, several proposals have been developed focused on modifying the concentrations of compounds of interest in microalgae, such as the one described in patent application US20100255458A1, in which the use of concentrators is mentioned. Compound parabolic as a strategy to improve the level of irradiance and improve the effective area of the light that falls on the crops grown in a photobioreactor. Said application indicates that the cultures may be of photoautotrophic organisms, but does not describe a particular type of organism. This invention describes automated management systems that allow the control of culture conditions. Likewise, it is described that the proposed system could be used in a system with lighting with lamps or outdoors. To control the temperature of the liquid medium, it describes the use of Peltier cells, but the number of cells or the power of these elements is not indicated, nor is the location of the cells in the system detailed. This invention does not indicate the flow of liquid medium, nor the location of the CPC and the photobioreactor. In the design proposed in the application cited above, the range of conditions in which the system can work is not indicated, nor is the type of photoautotrophic organisms that could be kept in the system. The description of the invention is very general and does not show specific data on the use of this type of system or the quality of the biomass produced.
En la patente US5958761A se describe un sistema tubular de f otobioreactor acoplado a un sistema concentrador parabólico compuesto (CPC) usado para optimizar la distribución de la luz. En el sistema se indica el uso de una bomba de intercambio de calor para controlar la temperatura del medio líquido. La temperatura del tubo se mantiene controlada por medio de un baño externo de agua a una temperatura de entre 27 a 32°C. El sistema descrito fue utilizado para la producción de betacaroteno y compara el rendimiento respecto a otros sistemas utilizados al exterior. En la descripción, no muestra las condiciones de operación y el tipo de organismo utilizado. Patent US5958761A describes a tubular photobioreactor system coupled to a compound parabolic concentrator (CPC) system used to optimize light distribution. The system indicates the use of a heat exchange pump to control the temperature of the liquid medium. The temperature of the tube is kept controlled by means of an external water bath at a temperature between 27 to 32°C. The described system was used for the production of beta-carotene and compares the performance with respect to other systems used abroad. In the description, it does not show the operating conditions and the type of organism used.
Finalmente en el Artículo de [Lopes, A., Santos F.M., Silva, T.E.C., Vilar V.J.P. Pires, J.C. 2020. Outdoor cultivation of the microalga Chlorella vulgaris in a new photobioreactor configuration: The Effect of Ultraviolet and Visible Radiation. Energies 13 (8) : 18 pp.1962] , se describe el uso de un sistema de cultivo de microalgas Chlorella vulgaris en un f otobioreactor tubular con un CPC bajo condiciones a cielo abierto. En este trabajo se investigó el efecto de la radiación ultravioleta y la luz visible respecto a la producción de biomasa y la capacidad de remoción de nutrientes por periodos de 80 horas. El sistema mantiene el control de la temperatura mediante un baño con termostato en valores entre 15°C y 35°C para permitir el crecimiento de Chlorella vulgaris . El sistema descrito en este Artículo se mantiene con una inclinación en un ángulo de 45°. En términos de producción de biomasa, la máxima tasa de crecimiento fue en promedio de 2 ± 0.6 xlO
en promedio con un nivel de radiación (UV y visible) entre 9 W m~2(45 pmol m~2 s~2) y 143 W nh2 (750 pmol nh2
s-1 ) . En este trabaj o de investigación con el uso de Chl orella vulgari s y el sistema de f otobioreactor tubular con CPC, no se describe la composición proximal y contenido de pigmentos de las células en el sistema de cultivo y, debido a que el CPC se mantuvo con un alto grado de trucamiento ( los autores no detallan el valor de truncamiento ) , los niveles de irradiancia máximos utili zados están en valores de 143 W
( 750 pmol m s-1 ) , por lo la producción de lipidos de las microalgas no se incrementa, conservándose las mismas características de un cultivo convencional . Finally in the Article by [Lopes, A., Santos FM, Silva, TEC, Vilar VJP Pires, JC 2020. Outdoor cultivation of the microalga Chlorella vulgaris in a new photobioreactor configuration: The Effect of Ultraviolet and Visible Radiation. Energies 13 (8) : 18 pp.1962], the use of a Chlorella vulgaris microalgae culture system in a tubular photobioreactor with a CPC under open air conditions is described. In this work, the effect of ultraviolet radiation and visible light on biomass production and nutrient removal capacity for periods of 80 hours was investigated. The system maintains temperature control by means of a bath with a thermostat at values between 15°C and 35°C to allow the growth of Chlorella vulgaris. The system described in this Article is kept tilted at an angle of 45°. In terms of biomass production, the maximum growth rate was on average 2 ± 0.6 xlO on average with a radiation level (UV and visible) between 9 W m~ 2 (45 pmol m~ 2 s~ 2 ) and 143 W nh 2 (750 pmol nh 2 s -1 ). In this research work with the use of Chl orella vulgari s and the tubular photobioreactor system with CPC, the proximal composition and content of pigments of the cells in the culture system are not described and, because the CPC remained with a high degree of truncation ( the authors do not detail the truncation value ) , the maximum irradiance levels used are at values of 143 W (750 pmol ms -1 ), so the production of lipids in microalgae does not increase, keeping the same characteristics of a conventional culture.
Ninguno de lo desarrollos propuestos permite obtener microalgas con un contenido de lipidos incrementado , ni mucho menos modi ficar la porporción entre los distintos tipos de lipidos presentes en las células de la microalga . Aunado a lo anterior, en todos los métodos para el cultivo de microalgas disponibles , las células mantienen sus pigmentos intactos , por lo que la biomasa obtenida, al ser utili zada en procesos de obtención de otros porductos , conserva los pigmentos f otosintéticos los cuales pueden quedar como contaminantes del producto final . None of the proposed developments makes it possible to obtain microalgae with an increased lipid content, much less modify the proportion between the different types of lipids present in the microalgae cells. In addition to the above, in all the methods for the cultivation of microalgae available, the cells keep their pigments intact, so that the biomass obtained, when used in processes to obtain other products, conserves the photo-synthetic pigments which can remain as contaminants in the final product.
En vista de problemática anterior, existe la necesidad de proporcionar un método para incrementar la concentración de lipidos en microalgas , que permita generar una biomasa rica en ácidos grasos , susceptible de ser usada en procesos de sintesis de otros productos tales como el biodiesel . Asimsimo , existe la necesidad de porporcionar un método para incrementar la concentración de lipidos en microalgas , que además permitan disminuir la concentración de pigmentos f otosintéticos , a fin de obtener células cloróticas de las microalgas , que requieran de menos proceso de puri ficación para poder ser utili zadas .
Sumario de la invención In view of the above problems, there is a need to provide a method to increase the concentration of lipids in microalgae, which allows the generation of a biomass rich in fatty acids, capable of being used in processes of synthesis of other products such as biodiesel. Likewise , there is a need to provide a method to increase the concentration of lipids in microalgae , which also allows to reduce the concentration of photosynthetic pigments , in order to obtain chlorotic cells from microalgae , which require less purification process to be able to be used. Summary of the invention
Con el fin de superar las limitantes de los métodos para el cultivo de microalgas , la presente invención tiene por obj etivo proporcionar un método para incrementar de forma controlada la cantidad de lipidos en células de la microalga Chl orella vulgari s que produzca células con baj o contenido de pigmentos f otosintéticos . In order to overcome the limitations of the methods for the cultivation of microalgae, the objective of the present invention is to provide a method to increase in a controlled manner the amount of lipids in cells of the microalga Chl orella vulgaris that produces cells with low o content of photosynthetic pigments.
Otro obj etivo de la presente invención es proporcionar un método para incrementar el contenido de lipidos de la microalga Chl orella vulgari s que permita obtener un perfil de lipidos adecuado para ser usadas como material precursor de la sintesis de biodiesel . Another objective of the present invention is to provide a method to increase the lipid content of the microalga Chl orella vulgaris that allows obtaining a suitable lipid profile to be used as precursor material for the synthesis of biodiesel.
Un obj etivo adicional de la presente invención, es proporcionar un método para incrementar el contenido de lipidos de cultivos de la microalga Chl orella vulgari s que esté adaptado para poder ser llevado a cabo baj o radiación solar directa sin afectar la producción de los compuestos de interés . An additional objective of the present invention is to provide a method to increase the lipid content of cultures of the microalga Chl orella vulgaris that is adapted to be carried out under direct solar radiation without affecting the production of the compounds of interest .
Otro obj etivo más de la presente invención, es proporcionar un método para incrementar el contenido de lipidos de la microalga Chl orella vulgari s capaz de proporcionar una biomasa rica en ácidos grasos de alto cetanaj e , libre de carotenos y baj o contenido de clorofila . Another objective of the present invention is to provide a method to increase the lipid content of the microalga Chl orella vulgaris capable of providing a biomass rich in high cetane fatty acids, free of carotenoids and low in chlorophyll content.
Otro obj etivo adicional de la presente invención, es proporcionar un método para incrementar el contenido de lipidos de la microalga Chl orella vulgari s que pueda mantener viable la biomasa aún baj o condiciones de muy alta irradiancia . Another additional objective of the present invention is to provide a method to increase the lipid content of the microalga Chl orella vulgaris that can keep the biomass viable even under conditions of very high irradiance.
Los obj etivos antes citados , asi como otros y las ventaj as de la presente invención, vendrán a ser aparentes de la siguiente descripción detallada de la misma .
Descripción de las Figuras de la Invención The aforementioned as well as other objects and advantages of the present invention will become apparent from the following detailed description thereof. Description of the Figures of the Invention
La figura 1 , muestra una vista en perspectiva del f otobiorreactor usado para llevar a cabo el método de la presente invención . Figure 1 shows a perspective view of the photobioreactor used to carry out the method of the present invention.
La figura 2 , muestra una gráfica en la que se observa el porcentaj e de truncamiento del concentrador de canal parabólico ( CPC ) usado en el f otobiorreactor empleado para llevar a cabo el método de la presente invención . Figure 2 shows a graph showing the truncation percentage of the parabolic trough concentrator (CPC) used in the photobioreactor used to carry out the method of the present invention.
Descripción detallada de la invención Detailed description of the invention
La presente invención proporciona un método para incrementar la cantidad de lipidos y de ácidos grasos en células de la microalga Chl orella vulgari s , que además disminuye la producción de clorofila, por lo que permite obtener material celular libre de pigmentos fotosintéticos , de manera que al ser destinado , por ej emplo , a la producción de biodiesel , se evita la contaminación del producto final , lo que mej ora la calidad del biodiesel obtenido y evita la acumulación de residuos en los motores que utilicen el biodiesel producido . Para lograr que las células de la microalga Chl orella vulgari s queden libres de pigmentos fotosintéticos , el método de la presente invención usa un concentrador Parabólico Compuesto ( CPC ) con una superficie reflectiva, asociado a un f otobiorreactor tubular, que permite concentrar la radiación incidente de manera eficiente de forma que se obtengan altos niveles de irradiancia sobre las células de la microalga en cultivo , en el orden de entre 400 hasta los 5000 pmol
que inducen una menor concentración de clorofila en las células de Chl orella vulgari s . Aunado a ello , el método de la presente invención permite mantener células viables de Chl orella vulgari s aún baj o las condiciones de la alta irradiancia lograda, sin necesidad de recurrir a
superficies de sombra para evitar el sobrecalentamiento del cultivo, usando celdas de Peltier colocadas de manera estratégica en el camino del flujo del cultivo. The present invention provides a method to increase the amount of lipids and fatty acids in cells of the microalga Chl orella vulgaris, which also decreases the production of chlorophyll, which allows obtaining cellular material free of photosynthetic pigments, so that at being destined, for example, to the production of biodiesel, contamination of the final product is avoided, which improves the quality of the biodiesel obtained and avoids the accumulation of residues in the engines that use the biodiesel produced. To ensure that the cells of the Chl orella vulgaris microalgae remain free of photosynthetic pigments, the method of the present invention uses a Compound Parabolic Concentrator (CPC) with a reflective surface, associated with a tubular photobioreactor, which allows concentrating the incident radiation. efficiently so that high levels of irradiance are obtained on the cells of the microalgae in culture, in the order of between 400 and 5000 pmol that induce a lower concentration of chlorophyll in Chl orella vulgari s cells. In addition to this, the method of the present invention allows to maintain viable Chl orella vulgari s cells even under the conditions of the high irradiance achieved, without the need to resort to shaded surfaces to prevent overheating of the culture, using Peltier cells strategically placed in the path of the culture flow.
Para lograr lo anterior el método de la presente invención comprende los siguientes pasos: a) adicionar un inoculo de células de la microalga Chlorella vulgaris Beyerinck en fase exponencial de crecimiento, a un medio de cultivo "f" [ (Guillard, R.R.L., Ryther, J.H. (1962) . Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Canadian Journal of Microbiology 8 (2 ) : 229-239) ] , preparado con agua dulce a pH de 8.0, hasta obtener una concentración de 450,000 células mi-1; b) colocar el cultivo de microalgas dentro de un f otobiorreactor tubular que tiene: un tubo de borosilicate (1) de 1.5 m de longitud y 7.75 cm de diámetro interno, colocado en el centro de una superficie reflectante de un concentrador parabólico Compuesto o CPCTo achieve the above, the method of the present invention comprises the following steps: a) add an inoculum of cells of the microalgae Chlorella vulgaris Beyerinck in exponential phase of growth, to a culture medium "f" [ (Guillard, RRL, Ryther, JH (1962).Studies of marine planktonic diatoms: I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran.Canadian Journal of Microbiology 8(2):229-239)], prepared with fresh water at pH 8.0, up to obtain a concentration of 450,000 cells mi -1 ; b) place the microalgae culture inside a tubular photobioreactor that has: a borosilicate tube (1) 1.5 m long and 7.75 cm in internal diameter, placed in the center of a reflecting surface of a Compound parabolic concentrator or CPC
(2) con un porcentaje de truncamiento del 70% y una razón de concentración geométrica de 2.11, estando dispuestos ambos con una inclinación de 15° y; un tubo de retorno(2) with a truncation percentage of 70% and a geometric concentration ratio of 2.11, both being arranged with an inclination of 15° and; a return pipe
(3) de 90 cm de longitud y 2.4 cm de diámetro con celdas de Peltier (4) de 6 amperes colocadas en su exterior, controladas por sensores de temperatura (5 y 6) dispuestos en la entrada y salida del tubo de borosilicato principal; c) hacer recircular el cultivo dentro del f otobiorreactor a 11 L min-1, por medio de una bomba de recirculación (7) , manteniendo el cultivo a una temperatura máxima de entre 33 y 35 °C usando las celdas de Peltier (4) ; d) mantener el cultivo iluminado en valores de irradiancia máximas de 5000 pmol s ' preferentemente por medio de radiación solar directa en un ciclo de luz y obscuridad
de por lo menos 4 dias; e) centrifugar la biomasa a entre 3800 y 4200 rpm, por 5 a 10 min a una temperatura de entre 4 °C y 6°C y; f) liofilizar la biomasa centrifugada a -50°C y a una presión de 0.11 bar para obtener células secas de Chlorella vulgaris libres de carotenos, con un perfil de ácidos grasos que produce un número de cetanos de entre 45 y 51, un contenido de clorofila-a de entre 1.5 y 2.9 pg cel-1, un contenido de clorofila-b de entre 1.33 y 2.9 pg cel-1 y, un contenido de clorofila-c de entre 1.17 y 5.26 pg cel-1. (3) 90 cm long and 2.4 cm in diameter with 6-amp Peltier cells (4) placed on the outside, controlled by temperature sensors (5 and 6) arranged at the inlet and outlet of the main borosilicate tube; c) recirculate the culture inside the photobioreactor at 11 L min -1 , by means of a recirculation pump (7), keeping the culture at a maximum temperature of between 33 and 35 °C using the Peltier cells (4) ; d) keep the culture illuminated at maximum irradiance values of 5000 pmol s 'preferably by means of direct solar radiation in a cycle of light and darkness of at least 4 days; e) centrifuge the biomass at between 3800 and 4200 rpm, for 5 to 10 min at a temperature between 4 °C and 6 °C and; f) lyophilize the centrifuged biomass at -50°C and at a pressure of 0.11 bar to obtain dry cells of Chlorella vulgaris free of carotenoids, with a fatty acid profile that produces a cetane number of between 45 and 51, a chlorophyll content -a of between 1.5 and 2.9 pg cel -1 , a chlorophyll-b content of between 1.33 and 2.9 pg cel -1 and a chlorophyll-c content of between 1.17 and 5.26 pg cel -1 .
En una modalidad preferida de la presente invención, el f otobiorreactor usado en el método de la presente invención, comprende además una válvula para desfogue de aire (8) que permite expulsar gases producidos durante el cultivo de las microalgas, un sensor de flujo másico (9) para el monitoreo del flujo del cultivo, una válvula para colecta de muestra de cultivo (10) y, una válvula para llenado del f otobiorreactor (11) . Asimismo, en una modalidad preferida de la presente invención el f otobiorreactor tiene 7 celdas Peltier para controlar la temperatura del cultivo. In a preferred embodiment of the present invention, the photobioreactor used in the method of the present invention also comprises an air vent valve (8) that allows gases produced during the cultivation of microalgae to be expelled, a mass flow sensor ( 9) for crop flow monitoring, a culture sample collection valve (10) and a photobioreactor filling valve (11). Likewise, in a preferred embodiment of the present invention, the photobioreactor has 7 Peltier cells to control the temperature of the culture.
Con el método antes descrito se obtiene una biomasa seca de células de Chlorella vulgaris con bajo contenido de pigmentos f otosintéticos y un alto contenido de ácidos grasos, que puede ser usada como precursor de biodiesel o bien ser usada en productos nutricionales , cosméticos o farmacéuticos. With the method described above, a dry biomass of Chlorella vulgaris cells with a low content of photosynthetic pigments and a high content of fatty acids is obtained, which can be used as a biodiesel precursor or used in nutritional, cosmetic or pharmaceutical products.
En la presente metodología, el uso de un concentrador parabólico compuesto integrado a un f otobiorreactor tubular, permite optimizar la distribución de la radiación solar en el cultivo de microalgas gracias a su diseño geométrico especifico que logra dirigir los haces de radiación incidente en el plano de apertura del concentrador hacia la superficie externa del tubo que contiene el cultivo.
Para el desarrollo de la metodología de la presente invención, se tomaron en cuenta varios parámetros de control importantes, uno de los cuales fue el regular la intensidad de la radiación disponible en el tubo que contiene las células de Chlorella vulgaris . Para ello, el diseño de la metodología se llevó a cabo mediante un estudio de truncamiento del Concentrador parabólico compuesto a usar como se muestra en la figura 2. En este caso, ya que se requerían niveles de irradiancia de entre 400 a 5000 pmol
, se analizaron diferentes niveles de truncado observando lo siguiente: para un concentrador sin truncar acoplado a un f otobiorreactor tubular de 8 cm de diámetro, la altura máxima fue de 88.7 cm y se alcanzó una razón de concentración geométrica de 2.5. Bajo esas condiciones, la radiación solar concentrada tuvo valores mayores a lo que soportan las microalgas (6190 pmol
s ) , por lo que a partir de esas dimensiones se modificó la altura para disminuir la razón de concentración geométrica hasta 2.1 con 26.6 cm de altura y de esta forma lograr las condiciones de radiación dentro del rango adecuado para el desarrollo de las células de Chlorella vulgaris . Los datos del análisis de truncamiento se muestran en la tabla 1. El porcentaje de truncamiento del Concentrador Parabólico compuesto fue de 70% y en ese punto la radiación solar concentrada fue de 5090 pmol In the present methodology, the use of a compound parabolic concentrator integrated into a tubular photobioreactor allows optimizing the distribution of solar radiation in the cultivation of microalgae thanks to its specific geometric design that manages to direct the incident radiation beams in the plane of opening of the concentrator towards the external surface of the tube containing the culture. For the development of the methodology of the present invention, several important control parameters were taken into account, one of which was to regulate the intensity of the radiation available in the tube containing the Chlorella vulgaris cells. For this, the design of the methodology was carried out through a truncation study of the compound parabolic concentrator to be used as shown in figure 2. In this case, since irradiance levels between 400 and 5000 pmol were required , different levels of truncation were analyzed observing the following: for an untruncated concentrator coupled to a tubular photobioreactor of 8 cm in diameter, the maximum height was 88.7 cm and a geometric concentration ratio of 2.5 was reached. Under these conditions, the concentrated solar radiation had values higher than those supported by microalgae (6190 pmol s ), so from these dimensions the height was modified to reduce the geometric concentration ratio to 2.1 with 26.6 cm height and thus achieve radiation conditions within the range suitable for the development of Chlorella cells vulgaris. The data of the truncation analysis are shown in table 1. The truncation percentage of the compound Parabolic Concentrator was 70% and at that point the concentrated solar radiation was 5090 pmol
Tabla 1. Relación entre las dimensiones del Concentrador Parabólico Compuesto (CPC) y la radiación incidente en el cultivo de microalgas. Table 1. Relationship between the dimensions of the Compound Parabolic Concentrator (CPC) and the incident radiation in the microalgae culture.
Porcentaje Altura Ancho Razón de Radiación de [m] [m] concentración solar truncamiento concentradaPercentage Height Width Radiation ratio of [m] [m] solar concentration truncation concentrated
— 2 —- two -
[ % ] [ pmo 1 m s[ % ] [ pmo 1 ms
0 0.8879 0.6303 2.50 61900 0.8879 0.6303 2.50 6190
20 0.7103 0.6276 2.49 608020 0.7103 0.6276 2.49 6080
30 0.6216 0.6206 2.46 6016
40 0.5328 0.6108 2.43 5920 30 0.6216 0.6206 2.46 6016 40 0.5328 0.6108 2.43 5920
50 0.444 0.5900 2.34 5713 50 0.444 0.5900 2.34 5713
60 0.3552 0.5664 2.25 5490 60 0.3552 0.5664 2.25 5490
70 0.2664 0.5314 2.11 5090 70 0.2664 0.5314 2.11 5090
80 0.1776 0.4868 1.93 4600 80 0.1776 0.4868 1.93 4600
Por otro lado, se analizó la concentración celular de los pigmentos f otosintéticos , de los lipidos y carbohidratos totales. En la tabla 2, se presentan los valores de la concentración de células, biomasa y de la composición proximal de los cultivos realizados de acuerdo con el método de la presente invención. On the other hand, the cellular concentration of photo-synthetic pigments, lipids and total carbohydrates were analyzed. Table 2 shows the values of cell concentration, biomass and proximal composition of the cultures made according to the method of the present invention.
Tabla 2. Composición proximal de los cultivos realizados
Table 2. Proximal composition of the cultures performed.
Tiempo Células PSO PST PC Lipidos , d ,í ,as Cel mi pq cel .-i pq cel .-i pq cel .-i pq cel .-i hidratos pg celTime Cells PSO PST PC Lipids, d,í,as Cel mi pq cel .-i pq cel .-i pq cel .-i pq cel .-i hydrates pg cel
0 450,000 137.78 145.19 7.41 8.67 14.260 450,000 137.78 145.19 7.41 8.67 14.26
1 450,000 133.83 150.12 16.30 11.78 15.121 450,000 133.83 150.12 16.30 11.78 15.12
2 560,000 102.38 115.48 13.10 10.09 9.182 560,000 102.38 115.48 13.10 10.09 9.18
3 389,000 131.39 146.82 15.42 10.03 8.163 389,000 131.39 146.82 15.42 10.03 8.16
4 260,000 203.88 212.51 8.63 50.00 6.614 260,000 203.88 212.51 8.63 50.00 6.61
5 310,000 159.34 196.85 36.51 17.93 7.445 310,000 159.34 196.85 36.51 17.93 7.44
6 496,000 126.95 131.21 4.25 8.36 8.95 6 496,000 126.95 131.21 4.25 8.36 8.95
Los datos muestran que la concentración de células en general tiene un decremento respecto al tiempo hasta el dia 4 de cultivo. En el dia 4 de cultivo se registra el máximo valor del peso seco orgánico (PSO) y peso seco total (PST) , pero con el mínimo valor del contenido de cenizas (PC) y el máximo valor de contenido de lipido. El contenido de carbohidratos tiende a disminuir conforme aumenta el tiempo de cultivo. The data shows that the cell concentration in general has a decrease with respect to time until day 4 of culture. On day 4 of culture, the maximum value of organic dry weight (PSO) and total dry weight (PST) is recorded, but with the minimum value of ash content (PC) and the maximum value of lipid content. Carbohydrate content tends to decrease as culture time increases.
En la tabla 3 se presentan los valores del contenido de pigmentos (Cío a: clorofila a; Cío b: clorofila b; Cío c: clorofila c y Car: carotenos) de Chlorella vulgaris sometidas al método de la presente invención, con respecto al tiempo.
Tabla 3. Contenido de pigmentos f otosintéticos Table 3 shows the pigment content values (Cío a: chlorophyll a; Cío b: chlorophyll b; Cío c: chlorophyll c and Car: carotenes) of Chlorella vulgaris subjected to the method of the present invention, with respect to time. Table 3. Content of photosynthetic pigments.
,, Cío a Cío b Cío c Car,, Cío a Cío b Cío c Car
Tiempo días , -i , -i , -i , -i pg cel pg cel pg cel pg cel Time days , -i , -i , -i , -i pg cel pg cel pg cel pg cel
0 7.18 2.22 0.00 1.69 0 7.18 2.22 0.00 1.69
1 5.82 3.06 2.19 0.18 1 5.82 3.06 2.19 0.18
2 1.31 1.34 1.17 0.00 2 1.31 1.34 1.17 0.00
3 1.05 1.33 1.57 0.00 3 1.05 1.33 1.57 0.00
4 2.90 4.66 5.26 0.00 4 2.90 4.66 5.26 0.00
5 2.37 2.92 4.36 0.00 5 2.37 2.92 4.36 0.00
6 2.33 2.18 2.20 0.00 6 2.33 2.18 2.20 0.00
Los datos obtenidos muestran que los contenidos de clorofila (a, b y c) disminuyen de forma continua respecto al tiempo hasta el dia 3 y a partir del dia 4 hay un ligero incremento en el contenido. El contenido de carotenos mostró un decremento del dia 0 al dia 1 y posteriormente se encontró en valores no detectables . The data obtained show that the contents of chlorophyll (a, b and c) decrease continuously with respect to time until day 3 and from day 4 there is a slight increase in content. The carotene content showed a decrease from day 0 to day 1 and later it was found in undetectable values.
Finalmente, para evidenciar la producción de ácidos grasos adecuados para ser usados como precursores de biodiesel, se concentró la biomasa de las células obtenidas por el método de la presente invención, por medio de una centrifuga a 4 °C y 4000 rpm por 10 min. El paquete de la biomasa de células se congeló a -80 °C hasta realizar la liof ilización a -50 °C y 0.11 kg cm2 de presión. Se pesó una alícuota de 50 mg de biomasa liofilizada y se realizó la extracción de lipidos por el método de Folch et al. (1957) , utilizando una solución de diclorometano-metanol (2:1) más BHT (Butilhidroxitolueno) al 0.01%. [Folch, J., Lee, M., Sloane-Stanley, G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissue. The Journal of Biological Chemistry, 22, 477-509] . La saponificación de los lipidos se realizó con una solución de 0.3 N de KOH metanólico al 90%. Una vez agregada la solución, se dejó en baño maria por 30 minutos a 60 °C. Cuando las muestras se encontraron a temperatura ambiente, se adicionaron 600 pl de agua y 400 pl hexano, para la extracción de los lipidos insaponif icables . La muestra colectada se acidificó con HC1 6N y
se agregó hexano para la separación de los lipidos saponif icables . La metilación de las muestras se realizó por el método de Metcalfe et al. (1966) [Metcalfe, L. D., Schmitz, A. A., Pelka, J. R. 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analytical Chemistry, 38 (3) , 514-515] y la composición de los ácidos grasos se analizó con un cromatógrafo de gases, con una columna capilar de 30 mm de longitud por 320 pm de diámetro, y grosor de la película de 0.25 pm, y un detector de flama ionizado, como gas de acarreo se utilizó hidrógeno. La identificación de los ácidos grasos se realizó por la comparación de los tiempos de retención con los del estándar, 37 "Component Supelco® FAME Mix SIGMA". Las cantidades de ácidos grasos de las muestras se calcularon por medio del programa MATLAB® en donde se relacionó la cantidad y el tipo de ácidos grasos obtenidos respecto a los del estándar. A partir del cultivo de Chlorella vulgaris obtenido por el método de la presente invención se obtuvo el perfil de los ácidos grasos de las células y se encontró que se produce un número de cetanos en valores en el rango de 45 a 51 y que están dentro de los valores reportados en las normas ASTM D6751 y EN 14214, de 47 y 51 descritas por Pandit et al. (2017) [Pandit, P. R., Fulekar, M. H., Karuna, M. S. L. 2017. Effect of salinity stress on growth, lipid productivity, fatty acid composition, and biodiesel properties in Acutodesmus obliquus and Chlorella vulgaris . Environmental Science and Pollution Research, 24 (15) , 13437-13451] . Por lo tanto, el perfil de ácidos grasos obtenidos corrobora que la biomasa producto del método de la presente invención, es susceptible de ser utilizada como precursora de la sintesis de biodiesel. Finally, to evidence the production of suitable fatty acids to be used as biodiesel precursors, the biomass of the cells obtained by the method of the present invention was concentrated by means of a centrifuge at 4 °C and 4000 rpm for 10 min. The cell biomass package was frozen at -80 °C until lyophilization at -50 °C and 0.11 kg cm 2 pressure. An aliquot of 50 mg of lyophilized biomass was weighed and lipid extraction was carried out using the method of Folch et al. (1957), using a solution of dichloromethane-methanol (2:1) plus BHT (Butylhydroxytoluene) at 0.01%. [Folch, J., Lee, M., Sloane-Stanley, GH 1957. A simple method for the isolation and purification of total lipids from animal tissue. The Journal of Biological Chemistry, 22, 477-509]. The saponification of the lipids was carried out with a solution of 0.3 N of 90% methanolic KOH. Once the solution was added, it was left in a water bath for 30 minutes at 60 °C. When the samples were at room temperature, 600 μl of water and 400 μl of hexane were added to extract the unsaponifiable lipids. The collected sample was acidified with 6N HC1 and hexane was added for separation of saponifiable lipids. The methylation of the samples was carried out by the method of Metcalfe et al. (1966) [Metcalfe, LD, Schmitz, AA, Pelka, JR 1966. Rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analytical Chemistry, 38 (3), 514-515] and the composition of the fatty acids was analyzed with a gas chromatograph, with a capillary column 30 mm long by 320 pm in diameter, and film thickness of 0.25 pm. , and an ionized flame detector, hydrogen was used as carrier gas. The identification of the fatty acids was carried out by comparing the retention times with those of the standard, 37 "Component Supelco® FAME Mix SIGMA". The amounts of fatty acids in the samples were calculated using the MATLAB® program, where the amount and type of fatty acids obtained were related to those of the standard. From the culture of Chlorella vulgaris obtained by the method of the present invention, the profile of the fatty acids of the cells was obtained and it was found that a number of cetanes is produced in values in the range of 45 to 51 and that they are within the values reported in the standards ASTM D6751 and EN 14214, of 47 and 51 described by Pandit et al. (2017) [Pandit, PR, Fulekar, MH, Karuna, MSL 2017. Effect of salinity stress on growth, lipid productivity, fatty acid composition, and biodiesel properties in Acutodesmus obliquus and Chlorella vulgaris. Environmental Science and Pollution Research, 24(15), 13437-13451]. Therefore, the profile of fatty acids obtained corroborates that the biomass product of the method of the present invention is capable of being used as a precursor for the synthesis of biodiesel.
La presente invención se ha descrito de acuerdo con una modalidad preferida; sin embargo, será aparente para un técnico con conocimientos medios en la materia, que podrán hacerse modificaciones a la invención, sin apartarse de su espíritu y alcance .
The present invention has been described according to a preferred embodiment; however, it will be apparent to a person skilled in the art that modifications may be made to the invention, without departing from its spirit and scope.
Claims
1.- Un método para obtener microalgas Chlorella vulgaris Beyerinck incoloras con alta concentración de lipidos caracterizado porque comprende los pasos de: a) adicionar un inoculo de células de la microalga Chlorella vulgaris Beyerinck en fase exponencial de crecimiento, a un medio de cultivo "f", preparado con agua dulce a pH 8.0, con una concentración de inoculo de 450,000 células mi-1; b) colocar el cultivo de microalgas dentro de un f otobiorreactor tubular que tiene: un tubo de borosilicato principal de 1.5 m de longitud y 7.75 cm de diámetro interno, dispuesto en el centro de una superficie reflectante de un concentrador parabólico Compuesto (CPC) con un porcentaje de truncamiento del 70% y una razón de concentración geométrica de 2.11, estando dispuestos ambos con una inclinación de 15° y; un tubo de borosilicato de retorno de 90 cm de longitud y 2.4 cm de diámetro con celdas de Peltier de 6 amperes colocadas en su exterior, controladas por sensores de temperatura dispuestos en la entrada y salida del tubo de borosilicato principal; c) hacer recircular el cultivo dentro del f otobiorreactor a 11 L min-1, por medio de una bomba de recirculación, manteniendo el cultivo a una temperatura máxima de entre 33 y 35 °C usando las celdas de Peltier; d) mantener el cultivo iluminado en valores de irradiancia máximas de 5090 pmol
s ; e) centrifugar la biomasa a entre 3800 y 4200 rpm, por 5 a 10 min a una temperatura de entre 4°C y 6°C y; f) liofilizar la biomasa centrifugada a -50°C y a una presión de 0.11 bar para obtener células secas de Chlorella vulgaris libres de carotenos, con un perfil de
ácidos grasos que produce un número de cetanos de entre 45 y 51, un contenido de clorofila-a de entre 1.5 y 2.9 pg cel-1, un contenido de clorofila-b de entre 1.33 y 2.9 pg cel-1 y, un contenido de clorofila-c de entre 1.17 y 5.26 pg cel-1. 1.- A method for obtaining colorless microalgae Chlorella vulgaris Beyerinck with a high concentration of lipids characterized in that it comprises the steps of: a) adding an inoculum of cells of the microalgae Chlorella vulgaris Beyerinck in exponential phase of growth, to a culture medium "f ", prepared with fresh water at pH 8.0, with an inoculum concentration of 450,000 cells mi -1 ; b) place the microalgae culture inside a tubular photobioreactor that has: a main borosilicate tube 1.5 m long and 7.75 cm in internal diameter, arranged in the center of a reflective surface of a Compound parabolic concentrator (CPC) with a truncation percentage of 70% and a geometric concentration ratio of 2.11, both being arranged with an inclination of 15° and; a return borosilicate tube 90 cm long and 2.4 cm in diameter with 6-amp Peltier cells placed on the outside, controlled by temperature sensors placed at the inlet and outlet of the main borosilicate tube; c) recirculate the culture inside the photobioreactor at 11 L min -1 , by means of a recirculation pump, keeping the culture at a maximum temperature of between 33 and 35 °C using the Peltier cells; d) maintain the illuminated culture at maximum irradiance values of 5090 pmol s ; e) centrifuge the biomass at between 3800 and 4200 rpm, for 5 to 10 min at a temperature between 4°C and 6°C and; f) lyophilize the centrifuged biomass at -50°C and at a pressure of 0.11 bar to obtain dry cells of Chlorella vulgaris free of carotenes, with a profile of fatty acids that produces a cetane number of between 45 and 51, a chlorophyll-a content of between 1.5 and 2.9 pg cel -1 , a chlorophyll-b content of between 1.33 and 2.9 pg cel -1 and a content of chlorophyll-c between 1.17 and 5.26 pg cel -1 .
2.- El método de acuerdo con la reivindicación 1, caracterizado porque la iluminación del paso d) se realiza preferentemente por medio de radiación solar en un ciclo de luz y obscuridad de por lo menos 4 dias. 2. The method according to claim 1, characterized in that the lighting of step d) is preferably carried out by means of solar radiation in a cycle of light and darkness of at least 4 days.
3.- El método de acuerdo con la reivindicación 1, caracterizado porque en el paso b) el f otobiorreactor comprende además una válvula para desfogue de aire (8) que permite expulsar gases producidos durante el cultivo de las microalgas, un sensor de flujo másico (9) para el monitoreo del flujo del cultivo, una válvula para colecta de muestra de cultivo (10) y, una válvula para llenado del f otobiorreactor (11) • 3. The method according to claim 1, characterized in that in step b) the photobioreactor further comprises an air vent valve (8) that allows gases produced during the cultivation of microalgae to be expelled, a mass flow sensor (9) for crop flow monitoring, a culture sample collection valve (10) and a photobioreactor filling valve (11) •
4.- El método de acuerdo con la reivindicación 1, caracterizado porque en el paso b) el número de celdas de Peltier es 7. 4. The method according to claim 1, characterized in that in step b) the number of Peltier cells is 7.
5.- Uso de las células secas de Chlorella vulgaris obtenidas por el método de acuerdo con la reivindicación 1, en la producción de biodiesel. 5.- Use of the dry cells of Chlorella vulgaris obtained by the method according to claim 1, in the production of biodiesel.
6.- Uso de las células secas de Chlorella vulgaris obtenidas por el método de acuerdo con la reivindicación 1, en la fabricación de productos nutricionales , cosméticos o farmacéuticos .
6. Use of the dry cells of Chlorella vulgaris obtained by the method according to claim 1, in the manufacture of nutritional, cosmetic or pharmaceutical products.
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| MX2020013590A MX2020013590A (en) | 2020-12-11 | 2020-12-11 | Method for obtaining colorless microalgae with a high concentration of lipids. |
| MXMX/A/2020/013590 | 2020-12-11 |
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| US5958761A (en) * | 1994-01-12 | 1999-09-28 | Yeda Research And Developement Co. Ltd. | Bioreactor and system for improved productivity of photosynthetic algae |
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| US20110275140A1 (en) * | 2007-01-17 | 2011-11-10 | Mccall Joe | Apparatus and methods for production of biodiesel |
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2020
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| US5958761A (en) * | 1994-01-12 | 1999-09-28 | Yeda Research And Developement Co. Ltd. | Bioreactor and system for improved productivity of photosynthetic algae |
| US20110275140A1 (en) * | 2007-01-17 | 2011-11-10 | Mccall Joe | Apparatus and methods for production of biodiesel |
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