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31/08/2011

PROGRAM OF SELECTION OF NEW SPECIES AND STRAINS OF MICROALGAES WITH HIGH POTENTIAL FOR LIPID PRODUCTION. Laura Carmona, Adelina de la Jara, Karen Freijanes, Patricia Assunção, Eduardo Portillo and Héctor Mendoza. de Biotecnología. División de Investigación y Desarrollo Tecnológico. Instituto Tecnológico de Canarias (ITC). Pozo Izquierdo, 35119 Sta. Lucía, Gran Canaria. *Contacts: lcarmona@itccanarias.org, depbio@itccanarias.org

Div./day

INTRODUCTION Of the 30.000 different species of microalgae, only 10% have been characterized and cultivated. The selection of new strains for biodiesel production or for other purposes is determined by the difficulty of characterization and selection (difficulty in sample processing, scalability, growth rate versus lipogenesis, etc.)

3,00

Grow rate

aDepartamento

2,50 2,00 1,50 1,00 0,50 0,00

In this project we develop a simple program (based on microculture and flow cytometry) for the selection of microalgae with high potential for lipids production. With this method we selected five strains, from 35 new field isolates and culture collections strains, that stood out for its high lipid production.

Correlation between cell growth rate and potential productivity

Strains

Growth rate (cell division/day)

(pg/cel.)

y = 1,6316x - 26,294 R² = 0,9147 1000 900 800 700 600 500 400 300 200 100 0

1,00E+11

1,00E+10

1,00E+09

1,00E+08

1,00E+07

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SSC

Strains

40

Correlation between dry weight and cell size (flow cytometry.)

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(FL2+FL3/SSC)

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Potential productivity values estimated by cytometric and automated cell counting. (SSC+FSC*Maximum density)/days)

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MATERIAL & METHODS

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The Techniques used: ` Microcultures: It can reproduce with some degree of reliability the growing conditions of the major massive cultures, without the constraints imposed by the need to handle large volumes. ` Multiparametric analysis by flow cytometry Flow cytometry is as a sensitive, specific and rapid technique that can be used to analyse at the same time a large number of cellular parameters (size, complexity, vitality, relative content of lipids, etc.) ` Conventional analytic: Cell growth, Dry Weight, Total Carbon and Nitrogen.

Strains

Potential productivity

Relative lipid content given by flow cytometry and Nile Red

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(µg/ml)/nºextr.

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Efficiency of extraction

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Relation between potential productivity and the efficiency of extraction

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Strains

Parameters for characterizing: ` Cell growth rate: Fundamental parameter not only of the potential productivity of the microalgae cultures, but also of the degree of adaptation of species and strains to the systems and production conditions. ` Cytometric indicator of dry weight per cell: The cytometric SSC parameter in combination with the rate of cell growth allows a rapid estimation of biomass production. ` Indicator of potential productivity: Estimated by flow cytometry and automated cell counting. (SSC+FSC*Maximum density)/days ` Relative lipid content: Cells stained with Nile Red and analyzed by flow cytometry. ` Ratio Carbon/Nitrogen: Indicator of the composition and physiological state of microalgae. ` Lipid extracts: The ability of extracting lipids from microalgae is so important as microalgae ability for producing them; moreover optimizes resources and reduces costs.

Extraction efficiency =fatty acids extracted (μg/ml) over the number of extractions performed

RESULTS & DISCUSION •These results demonstrate the full feasibility of developing a program of "screening" by microcultures and flow cytometry, making it possible to set cytometric markers for the selection of strains with high potential for growth and/or production of biodiesel. This is a fast and cheap screening method for the study of a large number of strains. •The increased scale of production systems does not change the outcome of the selection of strains and there is a significant correlation between the different growing methods: microcultures, photobioreactors and raceways. This result is of great interest since the microcultures management is easier and faster. Furthermore, the microcultures method do not have the environmental variations of the great outdoor culture systems, allowing a comparison between strains without the constraint of limiting parameters as temperature or light intensity. •The ability to extract lipids, is a decisive factor for a better selection of strains.

SCALABILITY: Relations between photobioreactor results and those obtained on culture chamber for the productivity and relative lipid content. Very significant correlation P = 0.99

Culture chamber

Cytometric PotencialProduction productivity

Photobioreactor

Finally, the data allowed the selection of the five following strains among the 35 studied for the biodiesel production on a large scale: •ITC 17(00)-S-0209 (Picochlorum sp.): It has a high growth rate, high lipid content and relative potential productivity. •ITC 04(00)-D.0209 (Chlorella sp.): It has also a high growth rate and productivity potential, as well as their high fat content . it is easy to extract the lipids. •ITC-ISO-01 (Isochrysis Galbana): It has a high relative content in lipids, high potential productivity and it is easy to extract the lipids. •ITC05(00)-D.0209 / ITC10(00)-D.0209 (Micractinium sp.): It has a high growth rate, relative content of lipids and potential productivity. • ITC03(00)-D.0209 (Scenedesmus sp.): It has a high growth rate, relative lipid and potential productivity.

Supported by PSE Microalgas (Proyectos Singulares Estratégicos) PSE-440000-2009-016

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Poster programe of selection of new species and strain