Biomass, lipid productivities and fatty acids composition of marine Nannochloropsis gaditana cultured in desalination concentrate

Bioresource Technology 197 (2015) 48–55

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Biomass, lipid productivities and fatty acids composition of marine Nannochloropsis gaditana cultured in desalination concentrate Ângelo Paggi Matos a,⇑, Rafael Feller b, Elisa Helena Siegel Moecke a,c, Ernani Sebastião Sant’Anna a a

Department of Food Science and Technology, Federal University of Santa Catarina, Av. Admar Gonzaga 1346, Itacorubi, 88034-001 Florianópolis, SC, Brazil Department of Chemical and Food Engineering, Federal University of Santa Catarina, Rua João Pio Duarte Silva, 241, Córrego Grande, 88037-000 Florianópolis, SC, Brazil c Laboratory of Environmental Engineering, Southern University of Santa Catarina, Av. Pedra Branca, Unidade Pedra Branca, 88137-270 Palhoça, SC, Brazil b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Higher Nannochloropsis gaditana

Desalination Concentrate

growth was observed at an optimum DC concentration of 75%.
The lipid content was 12.6% (w/w).
The DC concentration affects biomass, lipid productivity and fatty acids composition.
At optimum DC concentration, added glucose was the most favorable external carbon source.
Saturated fatty acids increased with increasing DC concentration.

a r t i c l e

i n f o

Article history: Received 3 July 2015 Received in revised form 3 August 2015 Accepted 4 August 2015 Available online 20 August 2015 Keywords: Microalgae Desalination wastewater Reuse of water Mixotrophic cultivation Biomass

Nannochloropsis gaditana culture

Golden lipids

a b s t r a c t In this study the feasibility of growing marine Nannochloropsis gaditana in desalination concentrate (DC) was explored and the influence of the DC concentration on the biomass growth, lipid productivities and fatty acids composition was assessed. The reuse of the medium with the optimum DC concentration in successive algal cultivation cycles and the additional of a carbon source to the optimized medium were also evaluated. On varying the DC concentration, the maximum biomass concentration (0.96 g L1) and lipid content (12.6%) were obtained for N. gaditana in the medium with the optimum DC concentration (75%). Over the course of the reuse of the optimum DC medium, three cultivation cycles were performed, observing that the biomass productivity is directly correlated to lipid productivity. Palmitic acid was the major fatty acid found in N. gaditana cells. The saturated fatty acids content of the algae enhanced significantly on increasing the DC concentration. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae have emerged as very promising candidates as a source material for the sustainable production of energy, offering significant advantages such as rapid growth, low demand for land area, and high yield of lipid- and carbohydrate-rich biomass per ⇑ Corresponding author at: Laboratory of Food Biotechnology, Federal University of Santa Catarina, 88034-001, Brazil. Tel.: +55 48 3721 5372. E-mail addresses: [email protected], [email protected] (Â.P Matos). http://dx.doi.org/10.1016/j.biortech.2015.08.041 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

acre (Baeyens et al., 2015; Foley et al., 2011; Stephens et al., 2010). Autotrophic growth is the most common approach to microalgae cultivation to provide algal biomass. However, to complement the commonly explored autotrophic activity, heterotrophic and mixotrophic algae systems have been considered as a viable alternative for supporting innovative bioprocesses (Ji et al., 2014; Perez-Garcia et al., 2011). Algal growth requires the availability of primary nutrients and micronutrients, which can be costly if they need to be added in large amounts. In this regard, nutrients from wastewater can be transferred to algal biomass, achieving simultaneously an

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economic microalgae production and efficient wastewater treatment (Pittman et al., 2011). For example, desalination through reverse osmosis (RO) is the most suitable procedure for obtaining fresh water in the semi arid region of Brazil, but this is associated with the problem of brine disposal. This residue, if not properly managed, can cause environmental problems such as the salination of agricultural land, which can render it infertile (Greenlee et al., 2009; Menezes et al., 2011). The brine discharge, known as desalination concentrate (DC), is predominately comprises mineral salts which include the cations Na+ (sodium), Ca+2 (calcium), Mg+2 (magnesium). With a growing need for the inland desalination of brackish water in Brazil, many solutions have been proposed for the use of DC as a source of nutrients, such as in aquaculture, the irrigation of halophyte plants, hydrophonic crops and, algaculture (Soares et al., 2006; Dias et al., 2010; Matos et al., 2015a). Additionally, an integrated scheme using DC for agricultural purposes (Tilapia + Spirulina + Atriplex) has been suggested by Sánchez et al. (2015), aimed at the production of human food and feed with high protein content for animals. Many species of microalgae are able to grow efficiently in wastewater conditions due to their ability to utilize the abundant organic and inorganic N and P in the wastewater. In addition, considering that microalgae require large amounts of water, the recycling of the water after harvesting can result in savings of up to 84% for water and 55% for essential nutrients such as nitrate and phosphate (Yang et al., 2011). Microalgae cultivation using desalination concentrate as a source of water and nutrients is of special interest in the valorization and recycling of brine waste loaded with valuable compounds (Moheimani et al., 2015). For example, Volkmann et al. (2008) reported the use of DC mixed with Paoletti medium for Arthrospira platensis cultivation aimed at food and feed production. Matos et al. (2015b) optimized a culture medium based on DC combined with Bold Basal Medium for freshwater Chlorella vulgaris cultivation. To complement these studies, it would be of great interest to grow marine algae with DC as a culture medium. Nannochloropsis is as a commonly cultivated organism which has been used to produce biofuel (Vooren et al., 2012), pharmaceutical compounds (Goiris et al., 2012; Yen et al., 2015), and fish feed (Camacho-Rodríguez et al., 2015). Nannochloropsis gaditana was selected for this study because of its high growth rate, high lipid productivity, and wide environmental tolerance (Griffiths and Harrison, 2009).

49

In this study, the use of desalination concentrate (DC) was investigated considering two main goals: (1) to evaluate the best DC concentration (in terms of percentage) for autotrophic growth of N. gaditana; and (2) to assess the influence of DC on biomass and, lipid productivities and fatty acids composition. Moreover, the reuse of DC wastewater, based on the optimum DC concentration for further autotrophic algal growth was tested and the effect of an additional carbon source on the biomass and lipid productivities was evaluated using the medium with the optimum DC concentration (Fig. 1). Based on the results, we hypothesis that marine algae N. gaditana can support high DC concentration than any other algae studied with higher biomass productivity and lipid content. 2. Methods 2.1. Algal strain and culture The marine species N. gaditana (clone 130) was kindly supplied by Banco de Micro-organismos Marinhos Aidar & Kutner (BMA&K) of the Oceanographic Institute at the University of São Paulo. The alga was maintained in autoclaved F/2 seawater medium (Table 1) suitable for marine algae (Guillard, 1975). 2.2. Desalination concentrate and experimental procedures Desalination concentrate (DC) was collected from an inland desalination plant, located in São João do Cariri, Paraíba, Brazil. The DC samples were collected in a 100-L plastic container and stored at 20 °C in the Laboratory of Food Biotechnology, Federal University of Santa Catarina, Santa Catarina, Brazil. The chemical composition of the DC (Table 1) was determined according to standard methods (APHA, 2005). The DC mixed with F/2 medium (based on seawater) in different concentrations (25%, 50%, 75% and 100% of DC) was used as the experimental media. A control test was conducted simultaneously using F/2 medium (0% DC). The initial N. gaditana concentration, approximately 105 cells mL1, was used for the preparation of the inoculum. All treatments were performed in 300-mL Erlenmeyer flasks and incubated at room temperature (25 ± 2 °C), sparing with saturated air-CO2, photoperiod of 12 h:12 h light/dark provided by fluorescent lamps, under a light intensity of 80 lmol m2 s1.

Fig. 1. Flow diagram for the cultivation of marine N. gaditana in desalination concentrate (DC) and subsequent studies using the DC percentage selected.

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Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55 Table 1 Compositions of desalination concentrate (DC) and F/2 medium. DC

Value ± means

F/2 medium

Value

pH Conductivity (mS cm1) TDS (mg L1) Cl (mg L1) Na (mg L1) CaCO3 (mg L1) 1 SO2 ) 4 (mg L Ca (mg L1) K (mg L1) 1 NO ) 3 -N (mg L Mg (mg L1) 1 + NH4 (mg L ) 1 PO3 ) 4 -P (mg L Fe (mg L1)

8.5 ± 0.5 4.5 ± 0.5 2190.5 ± 156.3 1691.3 ± 102 987.5 ± 99.5 985.2 ± 68.3 138.0 ± 3.2 126.5 ± 12.3 47.0 ± 0.2 30.0 ± 2.2 4.74 ± 0.8 1.35 ± 0.5 0.70 ± 0.1 0.13 ± 0.05

pH Conductivity (mS cm1) Stock solution (1.0 mL L1) NaNO3 (g L1) NaH2PO4H2O (g L1) Na2SiO39H2O (g L1) Trace metal solution (1.0 mL L1) FeCl36H2O (g L1) Na2EDTA2H2O (g L1) CuSO45H2O (g L1) Na2MoO42H2O (g L1) ZnSO47H2O (g L1) CoCl26H2O (g L1) MnCl24H2O (g L1) Vitamin solution (0.5 mL L1) Thiamine HCl (mg L1) Biotin (g L1) Cyanocobalamin (g L1)

8.0 ± 0.5 35.0 ± 0.5

Experimental cultures were obtained in duplicate over 7 days of cultivation. When green algae reached the stationary phase, samples were harvested by centrifugation at 4000 rpm for 20 min, washed with ammonium formate (Zhu and Lee, 1997) and centrifuged again (Nova Técnica, Piracicaba, Brazil). The algal pellet was transferred to a dish and dried in a dehydrator at 45 °C (Zhejiang, China) for lipid and fatty acid analysis. 2.3. Microscopic observation and visualization of lipid bodies The microalgal growth and intracellular lipid bodies were investigated by microscopic observation during the algal growth phase (5 and 7 days of cultivation) conducted with an inverted microscope (Nikon Eclipse Ti-S, Tokio, Japan), using 20 and 63 objectives. The intracellular lipid bodies of the algal cells were visualized using Nile Red stain (9-(diethyl amino) benzo [a] phenoxazin-5(5H)-one, Sigma–Aldrich), which was prepared as a stock solution of 250 mg L1 in acetone. One milliliter of green algae (grown for 14 days) was centrifuged at 4000 rpm for 10 min and a pellet was re-suspended in 1 mL of 20% DMSO. After vortexing for 10 min at room temperature, cells were centrifuged

75 5 30 3.15 4.36 9.8 6.3 22.0 10.0 180.0 200 1.0 1.0

at 4000 rpm for 10 min. The pellet was further suspended in 1 mL of water and vortexed before adding Nile Red strain (12.5 lL) and incubating for 5 min in the dark at room temperature (Ahmad et al., 2013). Stained cells were visualized using a Leica TCS-SP5 laser scanning confocal microscope (Wetzlar, Germany). Images were acquired using a 100 objective (HCX PL APO CS with a numerical aperture of 1.44 – oil immersion objective) with a Leica Type F immersion liquid. Images were taken with a Leica microsystem camera. The acquisition and processing of data were carried out using the LAS AF Lite software. 2.4. Culture medium recycling experiment To evaluate the influence of the recycling of the medium with the optimum DC concentration on the biomass and lipid productivity of N. gaditana, the medium was reused after harvesting by centrifugation for the next two cultivation cycles. The cultivation cycles were performed in duplicate using inverted conical photobioreactors with a working volume of 3.5 L (Fig. 2). In order to maintain satisfactory growth during three cultivation cycles, 3.0 L of the algal concentration was harvested by centrifugation for each

Fig. 2. Scheme showing the procedure to test the potential for the reuse of the water medium with a DC concentration of 75% in successive N. gaditana cultivation cycles.

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cultivation cycle, and a small portion (500 mL) of algal inoculum was kept inside the photobioreactor. Prior to a subsequent cultivation, the spent medium (supernatant) was re-introduced into the photobioreactors to proceed with the next cultivation cycle. For each cultivation cycle, the biomass was collected for lipid/fatty acid determination and 50 mL of the supernatant was recovered to determine the total nitrogen and total phosphorus content. The culture conditions (temperature, air, photoperiod and light intensity) were the same as those described in Section 2.2. Total nitrogen was determined through the persulfate digestion method on a Hach spectrophotometer (Loveland, USA) using PermachemÒ reagent kits. Total phosphorus was determined by the phosphorus (4500-P)/vanadomolybdophosphoric acid calorimetric method described in APHA (2005). 2.5. Mixotrophic cultivation on optimum desalination concentrate After the culture medium based on 75% DC concentration (optimum DC medium) had been established, N. gaditana was cultured mixotrophically using three different additional carbon sources: glucose (VetecÒ), glycerol (Nuclear) and glycerin (crude glycerin) with three different concentrations (1.0, 2.0 and 5.0 mL or g L1 depending on the substrate). All of the batch experiments were performed in 300-mL Erlenmeyer flasks applying 7 days of cultivation, under the conditions described in Section 2.2. 2.6. Determination of microalgal biomass The N. gaditana biomass concentration was estimated gravimetrically in terms of ash-free dry weight (AFDW) and also with the gravimetric method described by Zhu and Lee (1997). The biomass productivity (BP, g L1 day1) during the culture period was calculated from the equation:

BP ¼ ðX t  X 0 Þ=ðtx  t0 Þ

ð1Þ

where, Xt is the biomass production (g L1) at the end of the exponential growth phase (tx) and X0 is the initial biomass production (g L1) at t0 (day). The cellular lipid productivity was calculated as the product of BP and the fractional content (w/w) of the macromolecular pool in the biomass according to Dickinson et al. (2013): 1

Lipid productivity ðLP ; mg L1 d Þ ¼ Biomass productivity ðBP Þ  lipid=biomass ðw=wÞ

ð2Þ

2.7. Lipid extraction and fatty acids analysis Total lipids were extracted by the Soxhlet method with petroleum ether for 6 h, after acid digestion with 4 N HCl, followed by concentration in a rotary evaporator, drying in an oven and weighting (AOAC 996.06, 2005). The fatty acids composition was determined after conversion of the fatty acids to their corresponding methyl esters. The fatty acid methyl esters (FAME) were characterized on a gas chromatograph, model GC-2014 (Shimadzu, Kyoto, Japan), equipped with split-injection port, flame-ionization detector, a Restek a 105 m-long capillary column (ID = 0.25 mm) filled with 0.25 lm of 10% cyanopropylphenyl and 90% biscyanopropylsiloxane. Injector and detector temperatures were both 260 °C. The oven temperature was initially set at 140 °C for 5 min, and then programmed at 2.5 °C min1. The qualitative fatty acids composition was determined by comparing the retention times of the peaks with the respective fatty acids standards (Sigma, St. Louis, USA). The quantitative composition was obtained by area normalization and expressed as mass percent.

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2.8. Statistical analysis Statistical analysis was performed applying one-way analysis of variance (ANOVA), using STATISTICA Software (version 7.0) from StatSoft Inc. (2004). For all of the data analysis, a p-value of <0.05 was considered statistically significant. Where significant differences were observed, treatment means were differentiated using pairwise comparisons applying the Tukey test. 3. Results and discussion 3.1. Desalination concentrate Desalination concentrate (DC) is often rich in chlorides, sodium and calcium. In addition, other nutrients (nitrogen and phosphorus) and trace elements necessary for microalgae growth, including potassium, magnesium, and iron were all detected in DC (Table 1). Since DC has abundant mineral salts, the components existing in the DC were tested for advantageous microalgae growth. To assess the applicability of DC as a culture medium, the desalination waste was mixed with the F/2 medium before the algal cultivation (Fig. 1). In some cases, microalgae cannot grow normally in DC due to its high mineral salts strength. Similarly, C. vulgaris cannot grow normally in DC. For example, in a previous study by Matos et al. (2015b), the freshwater species C. vulgaris was highly inhibited using DC as culture medium. That was because the DC is abundant in sodium and chlorides associated with high salinity. In addition, it was detected in DC a small quantity of ammonia (1.35 mg L1) and sulfate (138.0 mg L1), some of which are toxic to aquatic life (Greenlee et al., 2009). On the other hand, marine microalgae N. gaditana performs well when exposure to a high DC concentration with higher biomass concentration and lipid content. These characteristics of DC suggested that it represents a good medium for marine N. gaditana growth in which can be replaced by conventional nutrients. 3.2. Effect of DC concentration on biomass and lipid content of N. gaditana The biomass concentration and lipid content of the experimental cultures with 0%, 25%, 50%, 75% and 100% of DC concentration are shown in Fig. 3. The microalgae cultured in 25% and 50% DC reached biomass concentrations of 0.63 and 0.81 g L1, respectively. In the control culture (0% DC), the biomass concentration was around 0.71 g L1. When N. gaditana was cultured in the 75% DC medium, the microalgae presented the maximum biomass concentration (0.96 g L1). However, algal growth was inhibited when the DC concentration was increased to 100%, and the biomass yield decreased considerably to about 0.33 g L1, but the lipid content remained relatively high, reaching 11.4% (w/w). Lipid accumulation to counterbalance the osmotic pressure has been previously reported (An et al., 2013), while lower N. gaditana biomass under 100% DC may be associated with an inability to maintain a satisfactory growth with only the mineral composition presented in DC (Table 1). The lipid content was observed to increase as the DC concentration increased from 25% to 75%, although a high lipid content was observed at under 75% DC (12.6% w/w), which is approximately treble that obtained for the control culture (4.7%) (as shown in Fig. 3). This result is consistent with previous reports that C. vulgaris has a higher lipid content when grown in desalination concentrate mixed with BBM medium (12.5%) than in 100% standard BBM medium (8.5%) (Matos et al., 2015a,b). However, the same authors observed that for optimum C. vulgaris growth, a maximum of 25% DC should be used. In contrast to the freshwater

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Fig. 3. Effect of concentration of desalination concentrate (DC) on biomass concentration (g L1) and lipid content (%).

alga C. vulgaris, the marine alga N. gaditana can easily adapt to exposure to a high DC concentration. Notably, the increases in the biomass concentration and lipid content of N. gaditana are much greater on exposure to high DC concentration. For this reason, and based on our observations (biomass concentration and lipid content) during the experimental procedures employing DC in the culture medium, we conducted the subsequent studies using a DC concentration of 75%. To visualize the N. gaditana cells under microscopic observation in the optimum medium containing a 75% DC concentration, the initial cultures (5 days) of algal cells were viewed under phasecontrast observation, as shown in Supplementary material Fig. S1 (a). After 7 days of cultivation, the algal cells appeared to be well adapted to the new proposed medium. As shown in Fig. S1(b), the marine N. gaditana cells were ellipsoidal with a diameter of 3–5 lm and, rich in chlorophyll. After staining with Nile Red, the bright yellow fluorescence from intracellular lipid globules and red fluorescence from chlorophyll could be observed (Fig. S1(c)). 3.3. Effect of the medium reuse on biomass and lipid productivities To study the effect of medium recycling on the performance of autotrophic N. gaditana culture, the optimum DC medium was reused until the total depletion of nutrients. The results for the biomass and lipid productivities of N. gaditana cultured autotrophically in the reused optimum DC medium are shown in Table 2. N. gaditana cells supported the spent medium for three cultivation cycles. The biomass accumulation during three cultivation cycles decreased as follows: 1st cycle = 1.06, 2nd cycle = 0.62 and 3rd

Table 2 Biomass and lipid productivities of N. gaditana cultured in medium with optimum DC concentration reused in successive cycles. Cultivation cycle

Biomass (g L1)

BP (mg L1 day1)

Lipid (%)

LP (mg L1 day1)

1st 2nd (1st reuse) 3rd (2nd reuse)

1.06 ± 0.2a 0.62 ± 0.1ab 0.14 ± 0.02b

151 ± 29a 88 ± 14ab 20 ± 2.5b

9.5 ± 0.8a 13.6 ± 0.7a 14.4 ± 1.0a

14.3 ± 2.2a 11.9 ± 1.7a 2.8 ± 0.4a

Data are means ± SD (n = 2). Each cycle represents 7 days of cultivation. Different letters in the same column correspond to significant differences (p < 0.05).

cycle = 0.14 g L1. A significant difference (p < 0.05) in the biomass concentrations for the three cultivation cycles was noted, since the total biomass concentration decreased considerably. On the other hand, the lipid contents of the microalgae cultivated in the recycled optimum DC medium showed a tendency toward an increasing with consecutive reuses of the recycled optimum DC medium. In addition, Fig. 4a shows the correlations between the lipid productivity (LP), biomass productivity (BP) and lipid content of N. gaditana for the three cultivation cycles performed. The biomass productivity appears to be directly correlated with the lipid productivity, but does not necessarily correlate with lipid content. Similar observations have also been reported by Griffiths and Harrison (2009), who noted that a high lipid content in microalgae does not necessarily represent high lipid productivity. In fact, the first cultivation cycle showed the highest biomass productivity (BP = 151 mg L1 day1) and lipid productivity (LP = 14.3 mg L1 day1). The second cultivation cycle presented lipid content (13.6%) higher than the first cultivation cycle (9.5%) while the highest lipid content (14.4%) was reached in the third cultivation cycle (Table 2). The result obtained in the third cultivation cycle is comparable with the study of Zhao et al. (2015) in N. gaditana Q6, but in disagreement with the results of Camacho-Rodríguez et al. (2015) and Mayers et al. (2014) involving Nannochloropsis sp. Considering that microalgae generally consume the majority of the nutrients, notably nitrogen and phosphorus, in the growth medium (Pittman et al., 2011), the concentrations of N and P in the reused optimum DC medium were measured. Park et al. (2011) mentioned that the N/P ratio in wastewater can ranged from 4/1 to 40/1 – N/P. The initial N and P ratio in optimum DC medium was 18.4 N/P (415.0 mg N/22.5 mg P), which was marginally higher than the theoretical ratio required for the growth of algae (16 N/P) (Redfield, 1958), illustrating that the N/P ratio in DC for N. gaditana growth is suitable. As can be seen in Fig. 4b, the initial N and P concentrations in the optimum DC medium were around 415 mg L1 total N and 22.5 mg L1 total P. In the first cultivation cycle, N. gaditana cells consumed 60% of the total N and 71.1% of total P available in the culture medium and after three cultivation cycles the corresponding values were 80% and 86%, respectively. Because of N and P was totally depleted, over the course of the cultivation cycles, the lipid content trend toward an enhancement with consecutive reuses of the recycled optimum DC medium. These results show that the reused optimum DC medium does not only support algal growth,

Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55

53

a

b

Fig. 4. Results for N. gaditana cultured in medium with the optimum DC concentration (75%) applied in three cultivation cycles. Each cycle involved 7 days of cultivation – 21 days in total. (a) Correlations between lipid productivity (LP), biomass productivity (BP) and lipid content of N. gaditana for successive cultivation cycles; (b) total nitrogen and total phosphate consumption by N. gaditana during the cultivation cycles.

Table 3 Comparison of biomass and lipid productivities of N. gaditana cultured in medium with optimum DC concentration, with additional carbon source in different concentrations (1.0, 2.0 and 5.0 g or mL L1) with 7 days of mixotrophic cultivation. Glucose (g L1)

Biomass (g L1)

BP (mg L1 day1)

Lipid (%)

LP (mg L1 day1)

0.72 ± 0.2a 1.10 ± 0.1a 0.95 ± 0.1a

102 ± 28a 156 ± 14a 135 ± 15a

4.8 ± 0.6a 5.0 ± 1.9a 5.5 ± 0.7a 8.7 ± 1.9a 1.0 ± 0.1a 1.3 ± 0.2a

Glycerol (mL L1) 1.0 0.42 ± 0.2a 2.0 0.15 ± 0.05a 5.0 0.10 ± 0.01a

60 ± 28a 21 ± 7a 2.5 ± 1a

6.2 ± 0.9a 3.9 ± 2.3a 3.8 ± 0.3a 0.8 ± 0.3a 11.0 ± 1.3a 0.3 ± 0.2a

Glycerin (mL L1) 1.0 0.19 ± 0.05a 2.0 0.25 ± 0.1a 5.0 0.22 ± 0.1a

27 ± 7a 35 ± 14a 31 ± 14a

9.0 ± 1.2a 2.4 ± 0.9a 12.0 ± 0.8a 4.3 ± 2.0a 8.5 ± 0.5a 2.6 ± 1.3a

1.0 2.0 5.0

Data are means ± SD (n = 3). Different letters in the same column correspond to significant differences (p < 0.05).

but also enhances the lipid content. Furthermore, the reused water and the mineral salts (N and P) present in the optimum DC medium were suitable for N. gaditana culture even after 3 cultivation cycles.

3.4. Effect of mixotrophic cultures on the biomass and lipid productivities Mixotrophic cultures of N. gaditana were conducted to investigate the biomass and lipid productivities under optimum DC medium. Glucose, glycerol and glycerin were used as substrates for microalgae cultivation. In this study, 2 g L1 of glucose supplementation produced 1.10 g L1 of biomass concentration and 8.7 mg L1 day1 of lipid productivity. This concentration of glucose was observed to be the most favorable carbon substrate and concentration for N. gaditana mixotrophic cultivation (Table 3). On using glycerol as the substrate, N. gaditana cells showed lower biomass and lipid productivity compared to glucose supplementation. When N. gaditana cells were grown on a glycerin substrate, high lipid content (8.5–12%) and low biomass concentration (0.19–0.25 g L1) were noted. Far higher rates of growth were obtained with the glucose substrate than the others tested, which included sugars, sugar alcohols and organic acids (Perez-Garcia et al., 2011). In our experiments with an additional carbon source, glucose could be considered a ‘‘preferred substrate” for mixotrophic N. gaditana cultivation because higher biomass and lipid productivities were noted. This could be because glucose possesses a high energy content per mol compared with other substrates (Boyle and Morgan, 2009).

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Table 4 Main fatty acids found in N. gaditana under different experimental conditions. Data are expressed as means ± SD (n = 2). Fatty acids (% TFAs) according to percentage of desalination concentrate (DC) in the medium C14:0 0% DC 25% DC 50% DC 75% DC 100% DC

6.5 ± 0.9 6.5 ± 1.1 5.5 ± 1.3 4.4 ± 0.6 5.5 ± 1.2

C16:0

C16:1

29.4 ± 1.1 44.5 ± 2.6 46.6 ± 2.8 48.7 ± 3.6 53.4 ± 2.3

30.7 ± 1.6 24.1 ± 1.4 24.4 ± 2.0 23.7 ± 1.8 22.2 ± 1.1

C18:0 0.5 ± 0.1 1.7 ± 0.2 1.4 ± 0.2 1.7 ± 0.4 2.4 ± 0.5

C18:1

C18:2

C18:3

C20:5n3

SFA

MUFA

PUFA

4.8 ± 0.6 9.3 ± 0.8 9.4 ± 1.2 12.5 ± 1.1 14.0 ± 0.7

3.3 ± 0.3 2.6 ± 0.4 2.0 ± 0.4 1.7 ± 0.1 0.5 ± 0.1

0.8 ± 0.1 0.5 ± 0.1 0.6 ± 0.2 0.6 ± 0.3 0.4 ± 0.1

12.2 ± 1.4 3.6 ± 0.8 4.2 ± 1.0 3.4 ± 0.5 0.2 ± 0.1

39.0 ± 2.5 56.6 ± 3.5 55.5 ± 4.1 56.9 ± 2.3 62.3 ± 2.4

41.5 ± 4.5 35.5 ± 2.6 34.6 ± 1.6 36.7 ± 2.1 36.4 ± 1.6

17.0 ± 2.4 7.5 ± 1.2 7.4 ± 1.0 7.0 ± 0.5 1.2 ± 0.1

51.2 ± 3.1 60.1 ± 2.7 54.4 ± 3.6

30.8 ± 2.7 28.3 ± 2.3 38.3 ± 3.1

16.3 ± 1.2 10.1 ± 1.0 6.0 ± 0.6

Fatty acids (% TFAs) obtained from cultures in the reuse of medium with optimum DC concentration 1st cycle 2nd cycle 3rd cycle

3.8 ± 0.9 4.2 ± 0.8 4.7 ± 0.9

40.2 ± 1.9 50.6 ± 2.8 45.6 ± 2.7

18.7 ± 1.4 12.9 ± 1.2 17.6 ± 1.3

2.7 ± 0.4 2.4 ± 0.2 1.9 ± 0.5

9.5 ± 1.1 13.7 ± 1.3 20.0 ± 1.7

5.5 ± 0.5 4.8 ± 0.6 2.7 ± 0.3

5.1 ± 0.9 4.2 ± 0.5 1.5 ± 0.2

5.7 ± 1.2 0.8 ± 0.1 1.2 ± 0.3

Fatty acids (% TFAs) profile under mixotrophic cultivation in medium with optimum DC concentration Glucose (1.0 g L1) (2.0 g L1) (5.0 g L1)

10.1 ± 0.8 10.4 ± 1.2 12.2 ± 1.4

50.5 ± 3.5 43.7 ± 2.5 41.5 ± 1.9

22.1 ± 1.5 23.2 ± 2.7 25.8 ± 3.1

1.8 ± 0.4 1.7 ± 0.3 1.5 ± 0.1

8.0 ± 1.2 7.9 ± 1.4 10.1 ± 1.1

0.6 ± 0.1 2.8 ± 0.4 2.2 ± 0.2

0.7 ± 0.1 1.0 ± 0.1 0.8 ± 0.1

0.8 ± 0.1 3.2 ± 0.3 3.3 ± 0.9

64.0 ± 4.1 59.8 ± 3.6 60.4 ± 3.6

9.7 ± 0.9 35.5 ± 1.9 35.9 ± 2.0

3.6 ± 0.3 7.0 ± 0.9 7.3 ± 1.0

Glycerol (1.0 mL L1) (2.0 mL L1) (5.0 mL L1)

4.8 ± 0.7 7.0 ± 1.1 6.3 ± 1.3

56.8 ± 3.7 54.0 ± 2.8 44.4 ± 3.6

14.2 ± 2.3 10.9 ± 1.9 11.1 ± 1.4

3.1 ± 0.3 5.0 ± 0.6 3.6 ± 0.4

9.6 ± 1.1 14.8 ± 1.7 22.0 ± 2.3

3.2 ± 0.7 2.2 ± 0.3 3.4 ± 0.2

0.8 ± 0.1 0.4 ± 0.1 0.5 ± 0.1

0.5 ± 0.1 0.8 ± 0.1 2.3 ± 0.3

63.3 ± 3.8 68.5 ± 5.3 67.5 ± 4.2

27.3 ± 2.6 26.8 ± 1.8 34.2 ± 2.2

5.9 ± 0.7 4.7 ± 0.3 9.3 ± 1.1

Glycerin (1.0 mL L1) (2.0 mL L1) (5.0 mL L1)

1.5 ± 0.3 1.3 ± 0.3 1.3 ± 0.2

47.9 ± 3.3 41.0 ± 3.2 39.5 ± 2.3

1.8 ± 0.2 1.9 ± 0.8 1.7 ± 0.5

17.3 ± 2.1 16.5 ± 2.4 15.6 ± 1.8

12.9 ± 1.2 24.1 ± 2.3 25.0 ± 1.1

0.7 ± 0.1 3.6 ± 0.4 2.2 ± 0.7

1.9 ± 0.3 1.9 ± 0.6 1.8 ± 0.3

1.6 ± 0.5 1.2 ± 0.6 1.1 ± 0.2

68.4 ± 3.5 60.5 ± 2.6 56.4 ± 2.2

21.2 ± 2.3 26.3 ± 2.3 26.7 ± 1.3

8.9 ± 1.0 9.6 ± 1.0 5.1 ± 0.8

3.5. Effect of experimental cultures on fatty acids composition of N. gaditana The fatty acids (FAs) composition of marine N. gaditana after applying the proposed experimental conditions is shown in Table 4. As a percentage of the total FA in N. gaditana, the predominant FAs for all DC concentrations in the medium were palmitic acid (C16:0; 29.4–53.4%), palmitoleic acid (C16:1, 22.2–30.7%) and oleic acid (C18:1, 4.6–14.0%), representing 81.1% of total FA content. Fatty acids present at moderate levels were eicosapentaenoic acid (C20:5n3, 0.2–12.2%) and myristic acid (C14:0, 4.4–6.5%), representing 15.5% of the total FA content. Fatty acids present at trace levels were linoleic acid (C18:2n, 0.5–3.3%) and linolenic acid (C18:3n, 0.4–0.8%), representing 3.4% of total FA contents. Algal FAs were highest in saturates (SFAs; 39.0–62.3%) followed by monounsaturates (MUFAs; 34.6–41.5%) and lowest polyunsaturates (PUFAs; 1.2–17.0%), when N. gaditana cells were cultivated media with increasing DC concentrations. It is evident that there is a tendency for N. gaditana cells to produce higher SFAs and lower PUFAs with increasing DC concentration in the culture medium (Table 4). For example, on increasing the DC concentration from 0% to 100%, the percentage of C16:0 increased from 29.4% to 53.4%, and the percentage of C20:5n3 dropped from 12.2% to 0.2%. In relation to the total FA content of the N. gaditana biomass after three cultivation cycles performed with the optimum DC medium, palmitic acid (40.2–50.6%) was predominant in all cycles (Table 4). During the first cultivation cycle the N. gaditana cells produced more PUFAs (16.3%), especially C20:5n3 acid (5.7% of total PUFAs). However, after the second cultivation cycle the algal cells were rich in SFAs (60.1%) and after the third cultivation cycle the biomass was abundant in MUFAs (38.3%). The data for the FAs obtained after three cultivation cycles of N. gaditana shown in Table 4, indicate that the reuse of the water, based on the optimum DC medium, could improve the diversity of the fatty acids composition of N. gaditana. Over the course of the reuse cycles with the optimum DC medium, PUFAs (C18:2, C18:3 and C20:5n3) have a tendency to decrease whereas the SFAs (C14:0 and C16:0) and MUFA (C18:1) have a tendency to increase.

Regarding the additional of a carbon source to the optimum DC medium, SFAs (mostly C16:0), accounted for approximately 39.5– 56.8% of the total FA content. Interestingly, on adding an extra carbon source (glucose, glycerol and glycerin) to the optimum DC medium, the production of SFAs decreased, while the percentage of PUFAs increased. In contrast, when N. gaditana cells were cultured in media containing increasing DC concentration and with limited nutrients (reuse of optimum DC water medium), the increase in SFAs may play a critical role in providing an appropriate degree of membrane fluidity for growth under ‘‘stress” condition. The additional of a carbon source to the optimum DC medium allows a shift in the metabolism of the algae resulting in more unsaturated (MUFAs + PUFAs) fatty acids being produced. 4. Conclusions Peaks in the biomass concentration (0.96 g L1) and lipid productivity (16.8 mg L1 day1) were observed using a DC concentration of 75%, indicating that this is the ideal concentration for the autotrophic growth of N. gaditana. The reuse of the optimum DC medium was supported by N. gaditana for three cultivation cycles. Increasing the DC concentration weakens the dominance of C20:5n3 (PUFA) and increases the percentage of C16:0 (SFA). These results indicate that DC is a suitable medium for N. gaditana cultivation and also demonstrate the valorization of desalination wastewater thought its application to algal mass production. Acknowledgments The authors would like to thank Dra. Flávia Marisa Prado Saldanha-Corrêa from the University of São Paulo for providing marine algal strain. We are also grateful for the laboratory facilities provided by the Laboratory of Analysis (LABCAL/UFSC) for this research. We would also like to acknowledge M.Sc. Eliana Medeiros de Oliveira for helping with the microscopy images. AP Matos is grateful to CAPES–Brazil for providing a doctoral scholarship.

Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55

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