Influence of desalinator wastewater for the cultivation of Arthrospira platensis . Fatty acids profile

En la región nordeste de Brasil, debido a la elevada salinidad de los acuíferos, existe la necesidad de utilizar procesos de desalación. Sin embargo, estos procesos generan residuos con alta concentración salina, con significativo impacto ambiental. El objetivo de este trabajo fue el cultivo de Arthrospira platensis en tres medios de cultivo diferentes: medio de Paoletti, medio de agua salinizada y medio de agua de desecho obtenida en el proceso de desalación de acuíferos. Las microalgas fueron cultivadas en laboratorio, con condiciones controladas, en fotobiorreactores de 4 L, a 30±1°C y periodos de 12 horas de claridad/oscuridad con iluminación de 140 μmol·m·s e inyección constante de aire (0,5 L·L·min). Fue verificado el efecto de los diferentes medios en la concentración celular, productividad, contenido total de lípidos y perfil de ácidos grasos. La mayor concentración celular, así como la productividad máxima fueron encontradas en el medio de agua de desecho de desalación, 4,954 (±0,554) g·L y 0,225 (±0,042) g·L·día respectivamente. En relación a los lípidos totales, se encontraron valores de 4,54% en el medio de agua de desecho de desalación y 4,69% en el medio de agua salinizada. En cuanto a los ácidos grasos, se obtuvieron altos índices de ácidos grasos saturados con ambos tratamientos. Con relación al ácido γ-linolénico, se encontró en un 13,09% en el medio de agua de desecho de desalación y en un 11,95% en el medio de agua salinizada.


INTRODUCTION
Large semi-arid areas of northeastern Brazil have serious problems with low pluviometric precipitations and seasonal droughts, besides a frequent occurrence of high levels of saline in its water sources.This problem can be minimized through the captation of groundwater and subsequent desalinization by reverse osmosis.However, the back-washing of membranes produces a highly salinated wastewater with a problematic destination.The present work aims to use this wastewater for composing a medium for Arthrospira platensis cultivation.The production of food and other compounds of interest from microalgae in non conventional systems present several advantages once it allows high levels of production, operating in controlled conditions and low levels of contamination.Arthrospira platensis is produced worldwide for obtaining proteins, carotenoids, vitamins, minerals and PUFAs (Polyunsaturated Fatty Acids), mainly γ-linolenic acid (Quoc and Dubacq, 1997;Xue et al., 2002;Hongsthong et al., 2003), being the major known source of vitamin B12 (Estrada et al., 2001;Duarte Filho et al., 2002).It presents compounds of pharmaceutical interest, having immuno-promoting effects, such as enhancing macrophage functions besides in vitro and in vivo antioxidant activity (Xue et al., 2002).Moreover, it is 85-95% assimilated by the organism, due to the lack of cellulose in its cell wall (Babadzhanov et al., 2004).Therapeutical significance of ω-3 e ω-6 PUFAs has been recently suggested through clinical and epidemiological investigations (Medina et al., 1998;Renaud et al., 2002;Kroes et al., 2003;Wen and Chen, 2003).Studies, mainly with γ-linolenic acid, suggest effects such as reduction in blood cholesterol, protection against some cancers, enhancement of the immune system, reduction of hyper lipidemia and obesity and partial inhibition of HIV-1 replication (Jiménez et al., 2003).These findings led to a great interest in the commercial development of a process for the production and extraction of these lipids.PUFAs can be obtained from animal and plant sources, being fish oil its main source.However, fish oils presents a production lower than the required demand, besides its unpleasant odor, contamination with heavy metals, presence of cholesterol, variable production and a complex fatty acid profile (Medina et al., 1998;Renaud et al., 2002;Kroes et al., 2003;Wen and Chen, 2003).On the other hand, PUFAs extracted from microalgae and other microorganisms lack these disadvantages and a simpler fatty acid composition facilitates purification (Medina et al., 1998;Zittelli et al., 1999;Wen and Chen, 2003).
Nevertheless, the PUFA content of algae depends not only on the species, but also on factors related to culture conditions (Volkman et al., 1989;Medina et al., 1998).Knowing that, the objective of this work was to evaluate the lipid and fatty acid contents of Arthrospira platensis grown in different media.

Microorganism and cultivation conditions
The Arthrospira platensis strain used in this work was given by the Laboratory of Biochemistry of Chemistry Department from the Federal University of Rio Grande Foundation -FURG/RS.It was kept in a Paoletti Synthetic Medium according to Ferraz et al. (1985), with modifications (Table 1).

Culture Media
Three different media were prepared for cultivation.Paoletti Synthetic Medium (PSM) was used as control medium.Salinated Water Medium (SWM), the second medium, was produced by adding 1.0 g•L -1 of NaCl to PSM and the third medium Desalinator Wasterwater Medium (DWWM) was produced by dissolving 50% of all components of PSM in the desalinization wastewater.This medium was produced as follows: after solubilization of the components in the wastewater, it was centrifuged at 4,000 rpm for 15 min and the precipitate was discarded.Desalinator wastewater was given by Reference Laboratory in Desalination, Campina Grande/PB, Brazil, and its composition is presented in Table 2, which was determined according to United States Environmental Protection Agency methods.
The pH of the media was adjusted to 9.4+0.2, with 3.0 M KOH solution using a potentiometer (Quimis, Q400A).

Inoculum preparation
Inocula of A. platensis were prepared for three media in 500 mL Erlenmeyer flasks.Microalgae were cultivated in 4 L photobiorreactors (working volume), with a photoperiod of 12 hours light/dark provided by fluorescent lamps (Philips, 20W) at a light intensity of 140 µmol•m -2 •s -1 and constant temperature of 30+1°C (climate controlled room).Experiments were initiated with 10% (v/v) of Micronutrients sol.(g•L -1 ): inoculum.Media agitation was carried out by a constant bubbling of air (0.5 L•L -1 •min -1 ).
The experiment ended with the decreasing of cell growth.At the end of cultivation, biomass was filtered (0.45 µm cellulose acetate filter), lyophylized (Terroni, LT 1000/8) and stored at -20°C.Specific growth rate and productivity were also determined.

pH determination
The pH of the growth media was measured at each 72 hours using a potentiometer.

Total lipid content and fatty acid profile
Total lipid content and fatty acid profile were determined only for SWM and DWWM.Lipids were extracted in chloroform:metanol (2:1) according to Folch et al. (1957) and quantified by gravimetry.
After lipid quantification, samples were esterified with boron trifluoride (1:10) as derivatizing agent according to Metcalfe et al. (1966).The methyl esters from fatty acids were identified on the basis of retention times corresponding to standards (Sigma Chemical Company).Identification was confirmed with Ackerman diagram.Quantification was performed by normalization of peak areas followed by estimation of percentages of each fatty acid in samples.
Gas chromatography of methyl esters was performed using a CG Varian Star (model 3400 CX) equipped with a flame ionization detector, using a DB-WAX capillary column (30 m ϫ 0.25 mm; 0.25 mm film thickness).The column was temperature programmed under the following conditions: initial temperature, 100°C; initial isotherm, 3 min; temperature increasing 5°C per min up to 180°C; intermediate isotherm, 1 min, then temperature increasing 1°C per minute up to 200°C; intermediate isotherm, 4 min, then temperature increasing 2°C per minute up to 210°C; intermediate isotherm, 3 min, then temperature increasing 5°C per minute up to 230°C; final isotherm, 12 min; injector and detector temperatures were 250 and 300°C, respectively.Helium was used as gas carrier (0.5 mL•min -1 ).

Statistical analysis
Results of the analyses were submitted to analysis of variance (ANOVA) with confidence level of 95% (p<0.05) in order to verify significant differences among media.Statistical analyses were carried out using Statistica ® 6.0 software.
Microalgae cultivated in DWWM presented lower productivity in the first days of cultivation, when compared with other treatments.However, it presented higher concentrations from day 10, reaching a maximum of 0.225 (±0.042) g•L -1 •day -1 at day 20 (Figure 2).Maximum values found for PSM and SWM were 0.148 (±0.019) g•L -1 •day -1 H. VOLKMANN, U. IMIANOVSKY, E. BADIALE FURLONG, J. L. BARCELOS OLIVEIRA AND E. SEBASTIÃO SANT'ANNA (day 6) and 0.165 (±0.020) g•L -1 •day -1 (day 9), respectively.Significant differences (p<0.05) were found between PSM and DWWM.The Salinated Water Medium did not present significant differences when compared to other treatments.Microalgae cultivated in DWWM presented higher productivity than those reported (0.175 g•L -1 •day -1 ) by Oliveira et al. (1999) but lower when compared to Travieso et al. (2001) who found a maximum productivity of 0.40 g•L -1 •day -1 for A. platensis cultivated in BG 11 medium.pH values ranged from 9.6 to 10.5, 9.6 to 10.6 and 9.4 to 10.2 for PSM, SWM and DWWM, respectively.This increase in pH can be correlated to the carbon source consumption.The bicarbonate ions are assimilated by A. platensis and subsequently converted into carbon dioxide and carbonate.During the first one is utilization in photosynthesis and excretion of ion carbonate into the medium; an increase in the pH of the system is generated due to the shift of the bicarbonatecarbonate equilibrium.

Effect of culture medium in lipidic content and fatty acids profile
Total lipid content was 4.54% and 4.69% in biomass cultivated in DWWM and SWM respectively, and did not present significant differences (p>0.05).These values are in agreement with those presented by Richmond (1990) but are lower than 6.96%, 6.38% and 7.09-8.03%reported by Oliveira et al. (1999), Xue et al. (2002) and Tokus , oglu and Ünal (2003), respectively.
Fatty acid profiles found in cells grown in DWWM and SWM presented 12 and 24 types of fatty acids, respectively (Table 3).Lauric (C12:0) and margaric (C17:0) acids were found in higher proportions in biomass of both cultivations, the sum of both representing 53.40% and 27.98% of total content of fatty acids from DWWM and SWM, respectively.The presence of margaric acid in A. platensis was reported by Xue et al. (2002) and Babadzhanov et al. (2004), but in contents of 0.1% and 1.2%, respectively.Studying forty different strains of A. platensis, Mühling et al. (2005) verified that 16 did not present the C17:0 fatty acid, indicating variability among them.Statistically significant differences were found between stearic (C18:0) and oleic (C18:1t) fatty acids.
A high content of saturated fatty acid was found in DWWM (57.69%) and in SWM (54.79%) (Figure 3).The saturated and monounsaturated fatty acids contents were higher in desalinator wastewater biomass while polyunsaturated content was higher in salinated water biomass.This can be due to the presence of C18:2t, C18:3 (α-linolenic), C20:2 and C22:6 (DHA) which were found only in the salinated water biomass.Tokus , oglu and Ünal (2003) showed a better distribution of fatty acids in these three groups presenting ranges from 36.40% to 39.29% of monounsaturated, 34.09% to 35.89% of saturated and 22.30% to 25.12% of polyunsaturated.Variations found in fatty acid profiles can be due to different or stressing conditions of cultivation.Walsh et al. (1997) mention an increasing in concentration of saturated fatty acids of microalgae under irradiance stress.
In conclusion, the data obtained in this work indicate that A. platensis cultivation in Desalinator Wastewater Medium is possible.It produced almost twice (91.49%) as much biomass as the control and presented expected lipid levels for this species.The presence of higher concentrations of saturated fatty acids and lower concentrations of γ-linolenic can be due to cultivation conditions.However, the use of this microalga in Desalinator Wastewater Medium large scale cultivations must be studied in order to determine its economical viability.

Table 1 Composition of Paoletti Synthetic Medium.
INFLUENCE OF DESALINATOR WASTEWATER FOR THE CULTIVATION OF ARTHROSPIRA PLATENSIS...