Bioresource Technology 197 (2015) 48–55

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

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
b
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

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). Nannochloropsis gaditana

The DC concentration affects biomass, culture
lipid productivity and fatty acids
composition. Golden lipids

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 a b s t r a c t

Article history: In this study the feasibility of growing marine Nannochloropsis gaditana in desalination concentrate (DC)
Received 3 July 2015 was explored and the influence of the DC concentration on the biomass growth, lipid productivities and
Received in revised form 3 August 2015 fatty acids composition was assessed. The reuse of the medium with the optimum DC concentration in
Accepted 4 August 2015
successive algal cultivation cycles and the additional of a carbon source to the optimized medium were
Available online 20 August 2015
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
Keywords:
(75%). Over the course of the reuse of the optimum DC medium, three cultivation cycles were performed,
Microalgae
Desalination wastewater
observing that the biomass productivity is directly correlated to lipid productivity. Palmitic acid was
Reuse of water the major fatty acid found in N. gaditana cells. The saturated fatty acids content of the algae enhanced
Mixotrophic cultivation significantly on increasing the DC concentration.
Biomass Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction acre (Baeyens et al., 2015; Foley et al., 2011; Stephens et al.,
2010). Autotrophic growth is the most common approach to
Microalgae have emerged as very promising candidates as a microalgae cultivation to provide algal biomass. However, to com-
source material for the sustainable production of energy, offering plement the commonly explored autotrophic activity, hetero-
significant advantages such as rapid growth, low demand for land trophic and mixotrophic algae systems have been considered as a
area, and high yield of lipid- and carbohydrate-rich biomass per viable alternative for supporting innovative bioprocesses (Ji et al.,
2014; Perez-Garcia et al., 2011).
Algal growth requires the availability of primary nutrients and
⇑ Corresponding author at: Laboratory of Food Biotechnology, Federal University
micronutrients, which can be costly if they need to be added in
of Santa Catarina, 88034-001, Brazil. Tel.: +55 48 3721 5372.
E-mail addresses: matos.a@posgrad.ufsc.br, angelosotam@gmail.com
large amounts. In this regard, nutrients from wastewater can be
(Â.P Matos). transferred to algal biomass, achieving simultaneously an

http://dx.doi.org/10.1016/j.biortech.2015.08.041
0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
 Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55
economic microalgae production and efficient wastewater treat-
ment (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 desali-
nation 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). Addition-
ally, 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, con-
sidering that microalgae require large amounts of water, the recy-
cling of the water after harvesting can result in savings of up to 84%
for water and 55% for essential nutrients such as nitrate and phos-
phate (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 environmen-
tal tolerance (Griffiths and Harrison, 2009).
Fig. 1. Flow diagram for the cultivation of marine N. gaditana in desalination concentrate (DC) and subsequent studies using the DC percentage selected.
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 concentra-
tion for further autotrophic algal growth was tested and the effect
of an additional carbon source on the biomass and lipid productiv-
ities 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 stan-
dard 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.
50 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 8.5 ± 0.5 pH 8.0 ± 0.5
Conductivity (mS cm1) 4.5 ± 0.5 Conductivity (mS cm1) 35.0 ± 0.5
TDS (mg L1) 2190.5 ± 156.3 Stock solution (1.0 mL L1)
Cl (mg L1) 1691.3 ± 102 NaNO3 (g L1) 75
Na (mg L1) 987.5 ± 99.5 NaH2PO4H2O (g L1) 5
CaCO3 (mg L1) 985.2 ± 68.3 Na2SiO39H2O (g L1) 30
1
SO2
4 (mg L ) 138.0 ± 3.2 Trace metal solution (1.0 mL L1)
Ca (mg L1) 126.5 ± 12.3 FeCl36H2O (g L1) 3.15
K (mg L1) 47.0 ± 0.2 Na2EDTA2H2O (g L1) 4.36
NO 3 -N (mg L
1
) 30.0 ± 2.2 CuSO45H2O (g L1) 9.8
Mg (mg L1) 4.74 ± 0.8 Na2MoO42H2O (g L1) 6.3
1
+
NH4 (mg L ) 1.35 ± 0.5 ZnSO47H2O (g L1) 22.0
1
PO3
4 -P (mg L ) 0.70 ± 0.1 CoCl26H2O (g L1) 10.0
Fe (mg L1) 0.13 ± 0.05 MnCl24H2O (g L1) 180.0
Vitamin solution (0.5 mL L1)
Thiamine HCl (mg L1) 200
Biotin (g L1) 1.0
Cyanocobalamin (g L1) 1.0

Experimental cultures were obtained in duplicate over 7 days of
cultivation. When green algae reached the stationary phase, sam-
ples were harvested by centrifugation at 4000 rpm for 20 min,
washed with ammonium formate (Zhu and Lee, 1997) and cen-
trifuged again (Nova Técnica, Piracicaba, Brazil). The algal pellet
was transferred to a dish and dried in a dehydrator at 45 °C (Zhe-
jiang, 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
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 productiv-
ity of N. gaditana, the medium was reused after harvesting by cen-
trifugation for the next two cultivation cycles. The cultivation
cycles were performed in duplicate using inverted conical photo-
bioreactors 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.
 Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55
cultivation cycle, and a small portion (500 mL) of algal inoculum
was kept inside the photobioreactor. Prior to a subsequent cultiva-
tion, 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 (opti-
mum 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 cultiva-
tion, under the conditions described in Section 2.2.
2.6. Determination of microalgal biomass
The N. gaditana biomass concentration was estimated gravimet-
rically 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 calcu-
lated from the equation:
BP ¼ ðX t  X 0 Þ=ðtx  t0 Þ
where, Xt is the biomass production (g L1) at the end of the expo-
nential 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
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 correspond-
ing 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% bis-
cyanopropylsiloxane. 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.
51
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 differ-
ences 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 phospho-
rus) 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 inhib-
ited using DC as culture medium. That was because the DC is abun-
dant 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 con-
tent. These characteristics of DC suggested that it represents a good
medium for marine N. gaditana growth in which can be replaced by
ð1Þ conventional nutrients.
3.2. Effect of DC concentration on biomass and lipid content of N.
gaditana
The biomass concentration and lipid content of the experimen-
tal 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, respec-
ð2Þ tively. 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 con-
centration (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 accumula-
tion 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 satisfac-
tory growth with only the mineral composition presented in DC
(Table 1).
The lipid content was observed to increase as the DC concentra-
tion 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
52 Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55

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 phase-
contrast 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 autotroph-
ically 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 Biomass BP Lipid (%)
cycle (g L1) (mg L1 day1)
1st 1.06 ± 0.2a 151 ± 29a 9.5 ± 0.8a
2nd (1st reuse) 0.62 ± 0.1ab 88 ± 14ab 13.6 ± 0.7a
3rd (2nd reuse) 0.14 ± 0.02b 20 ± 2.5b 14.4 ± 1.0a
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 recy-
cled optimum DC medium showed a tendency toward an increas-
ing with consecutive reuses of the recycled optimum DC medium.
In addition, Fig. 4a shows the correlations between the lipid pro-
ductivity (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 pro-
ductivity, 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 high-
est lipid content (14.4%) was reached in the third cultivation cycle
(Table 2). The result obtained in the third cultivation cycle is com-
parable 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 margin-
ally 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
LP 22.5 mg L1 total P. In the first cultivation cycle, N. gaditana cells
(mg L1 day1) consumed 60% of the total N and 71.1% of total P available in the
14.3 ± 2.2a culture medium and after three cultivation cycles the correspond-
11.9 ± 1.7a ing values were 80% and 86%, respectively. Because of N and P was
2.8 ± 0.4a
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

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 3.4. Effect of mixotrophic cultures on the biomass and lipid

Comparison of biomass and lipid productivities of N. gaditana cultured in medium productivities
with optimum DC concentration, with additional carbon source in different concen-
trations (1.0, 2.0 and 5.0 g or mL L1) with 7 days of mixotrophic cultivation.
Mixotrophic cultures of N. gaditana were conducted to investi-
Glucose Biomass BP Lipid (%) LP gate the biomass and lipid productivities under optimum DC med-
(g L1) (g L1) (mg L1 day1) (mg L1 day1) ium. Glucose, glycerol and glycerin were used as substrates for
1.0 0.72 ± 0.2a 102 ± 28a 4.8 ± 0.6a 5.0 ± 1.9a microalgae cultivation. In this study, 2 g L1 of glucose supplemen-
2.0 1.10 ± 0.1a 156 ± 14a 5.5 ± 0.7a 8.7 ± 1.9a tation produced 1.10 g L1 of biomass concentration and
5.0 0.95 ± 0.1a 135 ± 15a 1.0 ± 0.1a 1.3 ± 0.2a
8.7 mg L1 day1 of lipid productivity. This concentration of glu-
Glycerol (mL L1) cose was observed to be the most favorable carbon substrate and
1.0 0.42 ± 0.2a 60 ± 28a 6.2 ± 0.9a 3.9 ± 2.3a
concentration for N. gaditana mixotrophic cultivation (Table 3).
2.0 0.15 ± 0.05a 21 ± 7a 3.8 ± 0.3a 0.8 ± 0.3a
5.0 0.10 ± 0.01a 2.5 ± 1a 11.0 ± 1.3a 0.3 ± 0.2a On using glycerol as the substrate, N. gaditana cells showed lower
biomass and lipid productivity compared to glucose supplementa-
Glycerin (mL L1)
1.0 0.19 ± 0.05a 27 ± 7a 9.0 ± 1.2a 2.4 ± 0.9a tion. When N. gaditana cells were grown on a glycerin substrate,
2.0 0.25 ± 0.1a 35 ± 14a 12.0 ± 0.8a 4.3 ± 2.0a high lipid content (8.5–12%) and low biomass concentration
5.0 0.22 ± 0.1a 31 ± 14a 8.5 ± 0.5a 2.6 ± 1.3a (0.19–0.25 g L1) were noted. Far higher rates of growth were
Data are means ± SD (n = 3). obtained with the glucose substrate than the others tested, which
Different letters in the same column correspond to significant differences (p < 0.05). 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 mixo-
but also enhances the lipid content. Furthermore, the reused water trophic N. gaditana cultivation because higher biomass and lipid
and the mineral salts (N and P) present in the optimum DC productivities were noted. This could be because glucose possesses
medium were suitable for N. gaditana culture even after 3 a high energy content per mol compared with other substrates
cultivation cycles. (Boyle and Morgan, 2009).
54 Â.P Matos et al. / Bioresource Technology 197 (2015) 48–55

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 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:5n3 SFA MUFA PUFA
0% DC 6.5 ± 0.9 29.4 ± 1.1 30.7 ± 1.6 0.5 ± 0.1 4.8 ± 0.6 3.3 ± 0.3 0.8 ± 0.1 12.2 ± 1.4 39.0 ± 2.5 41.5 ± 4.5 17.0 ± 2.4
25% DC 6.5 ± 1.1 44.5 ± 2.6 24.1 ± 1.4 1.7 ± 0.2 9.3 ± 0.8 2.6 ± 0.4 0.5 ± 0.1 3.6 ± 0.8 56.6 ± 3.5 35.5 ± 2.6 7.5 ± 1.2
50% DC 5.5 ± 1.3 46.6 ± 2.8 24.4 ± 2.0 1.4 ± 0.2 9.4 ± 1.2 2.0 ± 0.4 0.6 ± 0.2 4.2 ± 1.0 55.5 ± 4.1 34.6 ± 1.6 7.4 ± 1.0
75% DC 4.4 ± 0.6 48.7 ± 3.6 23.7 ± 1.8 1.7 ± 0.4 12.5 ± 1.1 1.7 ± 0.1 0.6 ± 0.3 3.4 ± 0.5 56.9 ± 2.3 36.7 ± 2.1 7.0 ± 0.5
100% DC 5.5 ± 1.2 53.4 ± 2.3 22.2 ± 1.1 2.4 ± 0.5 14.0 ± 0.7 0.5 ± 0.1 0.4 ± 0.1 0.2 ± 0.1 62.3 ± 2.4 36.4 ± 1.6 1.2 ± 0.1

Fatty acids (% TFAs) obtained from cultures in the reuse of medium with optimum DC concentration
1st cycle 3.8 ± 0.9 40.2 ± 1.9 18.7 ± 1.4 2.7 ± 0.4 9.5 ± 1.1 5.5 ± 0.5 5.1 ± 0.9 5.7 ± 1.2 51.2 ± 3.1 30.8 ± 2.7 16.3 ± 1.2
2nd cycle 4.2 ± 0.8 50.6 ± 2.8 12.9 ± 1.2 2.4 ± 0.2 13.7 ± 1.3 4.8 ± 0.6 4.2 ± 0.5 0.8 ± 0.1 60.1 ± 2.7 28.3 ± 2.3 10.1 ± 1.0
3rd cycle 4.7 ± 0.9 45.6 ± 2.7 17.6 ± 1.3 1.9 ± 0.5 20.0 ± 1.7 2.7 ± 0.3 1.5 ± 0.2 1.2 ± 0.3 54.4 ± 3.6 38.3 ± 3.1 6.0 ± 0.6

Fatty acids (% TFAs) profile under mixotrophic cultivation in medium with optimum DC concentration
Glucose
(1.0 g L1) 10.1 ± 0.8 50.5 ± 3.5 22.1 ± 1.5 1.8 ± 0.4 8.0 ± 1.2 0.6 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 64.0 ± 4.1 9.7 ± 0.9 3.6 ± 0.3
(2.0 g L1) 10.4 ± 1.2 43.7 ± 2.5 23.2 ± 2.7 1.7 ± 0.3 7.9 ± 1.4 2.8 ± 0.4 1.0 ± 0.1 3.2 ± 0.3 59.8 ± 3.6 35.5 ± 1.9 7.0 ± 0.9
(5.0 g L1) 12.2 ± 1.4 41.5 ± 1.9 25.8 ± 3.1 1.5 ± 0.1 10.1 ± 1.1 2.2 ± 0.2 0.8 ± 0.1 3.3 ± 0.9 60.4 ± 3.6 35.9 ± 2.0 7.3 ± 1.0
Glycerol
(1.0 mL L1) 4.8 ± 0.7 56.8 ± 3.7 14.2 ± 2.3 3.1 ± 0.3 9.6 ± 1.1 3.2 ± 0.7 0.8 ± 0.1 0.5 ± 0.1 63.3 ± 3.8 27.3 ± 2.6 5.9 ± 0.7
(2.0 mL L1) 7.0 ± 1.1 54.0 ± 2.8 10.9 ± 1.9 5.0 ± 0.6 14.8 ± 1.7 2.2 ± 0.3 0.4 ± 0.1 0.8 ± 0.1 68.5 ± 5.3 26.8 ± 1.8 4.7 ± 0.3
(5.0 mL L1) 6.3 ± 1.3 44.4 ± 3.6 11.1 ± 1.4 3.6 ± 0.4 22.0 ± 2.3 3.4 ± 0.2 0.5 ± 0.1 2.3 ± 0.3 67.5 ± 4.2 34.2 ± 2.2 9.3 ± 1.1
Glycerin
(1.0 mL L1) 1.5 ± 0.3 47.9 ± 3.3 1.8 ± 0.2 17.3 ± 2.1 12.9 ± 1.2 0.7 ± 0.1 1.9 ± 0.3 1.6 ± 0.5 68.4 ± 3.5 21.2 ± 2.3 8.9 ± 1.0
(2.0 mL L1) 1.3 ± 0.3 41.0 ± 3.2 1.9 ± 0.8 16.5 ± 2.4 24.1 ± 2.3 3.6 ± 0.4 1.9 ± 0.6 1.2 ± 0.6 60.5 ± 2.6 26.3 ± 2.3 9.6 ± 1.0
(5.0 mL L1) 1.3 ± 0.2 39.5 ± 2.3 1.7 ± 0.5 15.6 ± 1.8 25.0 ± 1.1 2.2 ± 0.7 1.8 ± 0.3 1.1 ± 0.2 56.4 ± 2.2 26.7 ± 1.3 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%), repre-
senting 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 polyunsatu-
rates (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 pro-
duced 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 compo-
sition 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 car-
bon 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 cul-
tured 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 con-
centration of 75%, indicating that this is the ideal concentration
for the autotrophic growth of N. gaditana. The reuse of the opti-
mum DC medium was supported by N. gaditana for three cultiva-
tion 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
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2015.08.
041.
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