Lipids , fatty acids composition and carotenoids of Chlorella vulgaris cultivated in hydroponic wastewater By Fabiano

Medios de cultivo alternativos vienen siendo evaluados para el cultivo de microalgas, entre ellos, están los afluentes industriales y agrícolas, que posibilitan la reciclaje del residuo, bioconvirtiéndose en una biomasa enriquecida bajo el punto de vista nutricional, que puede ser utilizada como complemento alimenticio, para la acuacultura y en varias otras áreas de actuación. El presente trabajo tuvo como objetivo determinar los contenidos de lípidos, composición de ácidos grasos y carotenoides producidos por la microalga Chlorella vulgaris cultivada en solución hidropónica residual, con diferentes diluciones. Los resultados de los contenidos de lípidos totales no presentaron diferencia significativa. Los ácidos grasos predominantes fueron los 16:0, 18:0, 18:1 e 18:3n-6. Para los carotenoides totales, la dilución de la solución hidropónica residual no estimuló la producción de estos pigmentos por la microalga. La utilización de la solución hidropónica residual como medio de cultivo alternativo para Chlorella vulgaris genera buenas perspectivas para la producción de lípidos, ácidos grasos y carotenoides.


INTRODUCTION
An american called William Gericke suggested the word hydroponic to indicate the cultivation of plants in a liquid medium, making the cultivation of plants without soil popular in the 30´s (Jones Jr, 1982).However, the nutritive solution that feeds the plants, in the hydroponic technique, needs to be periodically replaced, offering a potentially nutritive residue solution.
Some micro algae present a larger fatty acid spectrum, when compared to oleaginous plants, containing a molecular structure with even more than 18 carbons (Belarbi et al., 2000).They generate good perspectives for oil production and may produce thirty times more oil than soybean per unit area.However, its major importance is related to the quality of the produced fatty acids, where most of them are composed of unsaturated fatty acids, like linolenic, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Skjak-Braek, 1992).
Prior studies showed fatty acid profile variations in microalgae species.This can be observed in response to different growth conditions (Fernández-Reiriz et al., 1989;Volkman et al., 1989;Dunstan et al., 1993).
The production of an algal biomass is not considered to be low cost.However, its profit compensates the investments, due to the value of some metabolites which can reach high prices on the international market (Borowitzka, 1990).
Fatty acids and carotenoids extracted from the microalgae can be offered with different purity contents, incorporated into other products or encapsulated, like in the docosahexaenoic acid market obtained from Crypthecodinium cohnii microalgae (Borowitzka, 1999).
Considering that the nutritive solution that feeds the plants in the hydroponic technique needs to be replaced periodically generating a kind of wastewater with macro and micronutrients in it, this experiment aimed at the evaluation of this wastewater as a potential cultivation medium to obtain lipids, fatty acids and carotenoids through Chlorella vulgaris microalgae.

Algal culture
The Chlorella vulgaris studied was donated by the Ficology Laboratory of the Federal University of São Carlos.The cultivation medium used in the maintenance of the microalgae was Bold's Basal Medium (BBM) which contained the following: mgL

Experimental installation
Cultures were kept under constant aeration, at a controlled temperature of 25±2 °C, pH 6,8 and continuous light of 150 μmol photon m -2 s -1 , coming from 40w fluorescent light bulbs.
A volume of 180mL, having a cellular density of 2.5x10 6 cellmL -1 of Chlorella vulgaris in BBM medium, was inoculated in inverted conic photobioreactors containing 3700 mL of four different cultivation media: BBM, which was considered as the reference cultivation; hydroponic wastewater (HW), diluted hydroponic wastewater 1:1 (50% residue to 50% deionized water) denominated as HW50 and diluted hydroponic wastewater 1:3 (25% residue to 75% deionized water) called HW25.To eliminate bacteria and protozoa, hydroponic wastewater was filtered and sterilized by autoclaving it for 20 min.Four independent repetitions were conducted for each treatment.The hydroponic wastewater was obtained from the Laboratory of Hydroponic of the Federal University of Santa Catarina, with its chemical characterization expressed in Table 1.

Analytical methods
The total content of Carotenoid was extracted from the fresh biomass with 90% acetone and determined by a spectrophotometric method according to the methodology developed by Strickland and Parsons (1972).
The cellular density was determined by counting the number of cells with light microscopy using a Neubauer Hematocytometer.
The lipid determination was done according to the methodology described by Bligh and Dyer (1959), where solvents used in the extraction were chloroform-methanol in a proportion of 1:2 (v/v).
From the extract obtained in the total lipid determination, the transesterification of the fatty acids was done according to the procedures developed by Hartman and Lago (1973).Fatty acid methyl esters were analyzed by gas chromatography with a flame ionization detector (Varian Star 3400CX), nitrogen was used as the carrier gas in a fused-silica capillary column DB-WAX (J&W Scientific) 30 m long and with a 0.32 mm inner diameter with an immobilized polyethylene glycol film thickness of 0.25 μm.
The operational conditions programmed were: initial column temperature of 50 °C for 2 minutes, elevated to 250 °C at a rate of 10 °C per minute, kept at the final temperature for 20 minutes.The temperature of the detector was 300 °C and the injector had a temperature of t 250 °C.Fatty acids were identified through the comparison of time retention with standards (Supelco).

Statistics Analysis
The statistics analysis was done using the variance analysis (ANOVA) and Tukey test, in a confidence level of 95% (p < 0.05), in order to show the differences between the means of each treatment.

RESULTS
Table 2 shows the results of the lipid and carotenoid contents produced by Chlorella vulgaris microalgae in different media after seven days of cultivation.
Although lipid contents did not show any significant differences (p > 0.05), the microalgae cultivated in HW25 presented the highest content among the other hydroponic wastewater solutions.
In a comparison of total carotenoid values, it was observed that the HW and HW50 cultivations presented higher contents, being significantly different (p < 0.05) from BBM and HW25 cultivations.While the concentration of total carotenoids produced in HW25 showed lower contents among the microalgae cultivated in hydroponic wastewater, this treatment did not show significant difference (p > 0.05) in relation to the control culture (BBM), indicating the feasibility of carotenoid production using this culture medium.
Table 3 shows Chlorella vulgaris, fatty acid composition after seven days of cultivation in different media The fatty acids 16:0, 18:0, 18:1 and 18:3n-6 were the ones found in larger concentrations.The other fatty acids were presented in smaller quantities, except for 20:0 from HW treatment and fatty acid 20:5 cultivated in HW25.
The percentage of saturated fatty acids of HW25 cultivation showed a significant difference (p < 0.05) when compared to HW and HW50 cultivations.It showed that the microalgae cultivated in HW25 presented a lower quantity of saturated fatty acids.On the other hand, it was the one that had the highest content of unsaturated fatty acids, as well as polyunsaturated fatty acids, but did not show significant difference (p > 0.05), except for HW.

DISCUSSION
The dilution of the hydroponic wastewater causes the reduction of important nutrients, like nitrogen and phosphorus.This nutrient deficiency causes lipids to be preferentially synthesized, resulting in a higher accumulation of fat in the algae cells (Rigano et al., 1998).With nitrogen reduction in the medium, a diminishing of the amino acid quantity in the cell occurs, consequently increasing the percentage of lipids (Mandalam and Palsson, 1998).
In another study done by Santos et al. (2003), it was observed that lipid contents decrease in Spirulina maxima microalgae from 23.16 to 8.35% with a drastic increase in nitrogen concentration from zero to 2.5gL -1 .However, there was a lower variation in lipid content when the nitrogen concentration remained between zero and 0.2gL -1 .Piorreck et al. (1984) observed that nitrogen concentration diminishing in a green algae medium, including Chlorella vulgaris, lead to a lipid increase, reaching approximately 45%.
Lipid production and accumulation in microalgae involve specific factors for each species and cultivation conditions.Nevertheless, based on the results found in the literature, it is suggested that lipid content increases due to the limiting factor of nitrogen levels.
Although the results have shown that the lipid contents increased as hydroponic wastewater was diluted, they did not present any significant difference (p > 0.05) among treatments.This indicates that the metabolism of Chlorella vulgaris was not significantly affected by lipid synthesis when cultivated in this medium.
The total carotenoid contents in Chlorella vulgaris are coherent with the ones found in the literature, where values vary from 0.03 to 0.08 pgcell -1 in a study done by Rioboo et al. (2002).It was observed that hydroponic wastewater dilution unstimulated this pigment production by microalgae.Studies show that nitrogen makes the accumulation of carotenoid viable in some microalgaes (Del Campo et al., 2000).
We can conclude that the use of hydroponic wastewater as a Chlorella vulgaris alternative culture medium generates potential perspectives for the production of lipids, fatty acids and carotenoids.