Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate

Bioresource Technology xxx (2016) xxx–xxx

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Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate Ângelo Paggi Matos a,⇑, Monnik Gandin Cavanholi a, Elisa Helena Siegel Moecke a,b, Ernani Sebastião Sant’Anna a b

Department of Food Science and Technology, Federal University of Santa Catarina, Av. Admar Gonzaga 1346, Itacorubi, 88034-001 Florianópolis, SC, Brazil 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


Optimal 75% DC concentration is

Biomass prod.

Biomass and lipid productivities (mg L-1 day-1)

160

appropriate medium for N. gaditana cultivation.
DC provided a higher lipid productivity than the control medium.
The optimal photoperiod cycles for enhancing algal biomass were determined.
The use of the optimum DC medium was evaluated in nine treatments.
Higher SFA production was observed under autotrophic than heterotrophic condition.

a r t i c l e

a b s t r a c t

i n f o

Article history: Received 9 September 2016 Received in revised form 31 October 2016 Accepted 2 November 2016 Available online xxxx Keywords: Inland desalination wastewater Microalgae Photo-osmotic Lipid enhancement Saturated fatty acids

Lipid prod.

Conductivity

40

140

35

120

30

100

25

80

20

60

15

40

10

20 0

5 0%

25%

50%

75%

100%

AUTOTROPHIC

MIXOTROPHIC

HETEROTROPHIC

Conductivity (mS cm-1)

a

0

Desalination concentrate (DC)

This study investigated the cultivation of the marine alga Nannochloropsis gaditana in a medium based on desalination concentrate (DC) with an optimal concentration of 75% DC, under three trophic conditions and four photoperiod schedules. N. gaditana produced a peak biomass concentration (1.25 g L1) under mixotrophic culture condition and a photoperiod of 16L:08D. N. gaditana cells compensate to different light-dark regimes producing different amounts of protein (17.9–44.8%). The intracellular lipid content in N. gaditana cells increased both under autotrophic conditions with a 16L:08D cycle (16.7%), and under mixotrophic conditions with a 08L:16D cycle (15.7%). In heterotrophic culture, N. gaditana cells were rich in polyunsaturated fatty acids (46.0%). This study demonstrates an alternative approach to enhancing intracellular lipid content of the marine alga N. gaditana by modifying the photoperiod, trophic conditions and stress-salinity-conductivity with the use of a DC-based medium. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction

⇑ Corresponding author at: Laboratory of Food Biotechnology, Federal University of Santa Catarina, 88034-001, Brazil. E-mail addresses: [email protected], [email protected] (Â.P Matos).

Algal biodiesel is a third generation biofuel which shows potential for the sustainable production of fuel with a small footprint, and it offers reduced competition with food crops (Carioca et al., 2009). Although microalgal cultivation does not require arable land, it is associated with a high demand for large quantities of

http://dx.doi.org/10.1016/j.biortech.2016.11.004 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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Â.P Matos et al. / Bioresource Technology xxx (2016) xxx–xxx

water, fertilizers and solar radiation, which considerably limits the availability of suitable locations (Varshney et al., 2015). A series of strategies is being developed to circumvent these issues, such as the use of marine species (Simionato et al., 2011), cultivation coupled with wastewater treatment plants (Dickinson et al., 2013; Mahapatra et al., 2014; Pittman et al., 2011), and mixotrophic cultivation using readily available low cost or industrial wastederived carbon sources (Perez-Garcia et al., 2011; Selvaratnam et al., 2015). Different carbon sources have been evaluated for mixotrophic or heterotrophic microalgal cultivation, including, glucose (Mallick et al., 2011), acetate (Isleten-Hosoglu et al., 2012), glycerol (Abad and Turon, 2012), xylose (Leite et al., 2016) and urban wastewater (Henkanatte-Gedera et al., 2015), and all have generally been shown to increase intracellular lipid content. The most common procedure for the cultivation of microalgae is autotrophic growth. Under autotrophic cultivation the cells harvest light energy and use CO2 as a carbon source. A feasible alternative for autotrophic cultures, although restricted to a few microalgal species, is the use of their heterotrophic growth capacity in the absence of light, replacing the fixation of atmospheric CO2 by autotrophic cultures with organic carbon sources dissolved in the culture medium (Perez-Garcia et al., 2011). The mixotrophic growth regime is a variant of the heterotrophic growth regime, where CO2 and organic carbon sources are simultaneously assimilated and both respiratory and photosynthetic metabolism operate concurrently (Ji et al., 2014). Considering that microalgal species are not truly mixotrophs, although they have the ability to switch between phototrophic and heterotrophic metabolisms, Nannochloropsis is commonly cultivated under either mixotrophic (Cheirsilp and Torpee, 2012) or heterotrophic conditions (PerezGarcia et al., 2011). In order to exploit microalgae for biofuel production, a better understanding of the parameters influencing the growth rate and the biomass and lipids accumulation is critical to optimize the productivity (Griffiths and Harrison, 2009). As in the case for all photosynthetic organisms, light is a major factor to be considered, since it provides all the energy that supports metabolism. The capacity for an effective and prompt acclimation is therefore an important parameter to be considered in the characterization of an algal species for biomass production (Simionato et al., 2011). The marine species Eustigmatophyceae Nannochloropsis gaditana was selected for this study because of its high growth rate and lipid productivity, as well as its wide environmental tolerance (Selvakumar and Umadevi, 2014; Mitra et al., 2015). Furthermore, the marine algae of the Nannochloropsis genus are able to synthesize high value chemicals such as proteins and fatty acids with medium (C16 and C18) and long (C20:5ɷ3) chain carbons, which are ideal lipid sources for the production of the nutraceutically valuable eicosapentaenoic acid (EPA) and biodiesel (Matos et al., 2016a; Mitra et al., 2015; Tibbetts et al., 2015). Several recent studies have analyzed the growth of microalgae under a variety of wastewater conditions, in particular growth in multiple municipal, industrial and agricultural wastewaters (Pittman et al., 2011; Dickinson et al., 2013). These studies have mainly been focused on evaluating the ability of algae to remove N and P, and in some instances metals, from wastewater. However, more recently studies have extended the feasibility of algal systems in combination with desalination concentrate (DC) for simultaneous N and P removal with biomass and lipid production (Volkmann et al., 2008; Sánchez et al., 2015; Matos et al., 2016b). Furthermore, DC is abundant in inorganic minerals, such as Cl, Na+ and Ca+2, which can lead to salt stress in microalgae cultivation, especially due to the effect of salinity on biochemical composition (Volkmann et al., 2008). For example, Matos et al. (2014) cultivated freshwater Chlorella vulgaris in dilutions of 25%, 35%, 45% and 55% DC mixed with Bold Basal Medium (BBM) and demon-

strated that incubating C. vulgaris with the higher DC concentrations of 35–55% resulted in growth-salt stress accompanied with lower biomass concentration and lipid production in comparison to control (BBM). According to Kumar et al. (2015), the salt concentration influences algae via effects on osmotic stress, salt stress, and cellular ionic ratios. For marine algae, salinity is expected to have some effect on the metabolism, especially on important cell membrane constituents, such as lipids (Takagi and Karseno, 2006) and fatty acids composition (An et al., 2013). In this context, cells of the marine alga N. gaditana were successfully cultured in an optimized 75% DC medium under two-stage photoautotrophicmixotrophic conditions using glucose (2.0 g L1) as a carbon source (Matos et al., 2015). In addition, N. gaditana has a tendency toward the synthesis of higher saturated fatty acids (C16:0 palmitic and C18:0 stearic acids) when increasing DC concentrations in the culture medium. Hence, the choice of marine N. gaditana in this study was motivated by its metabolic versatility that includes the ability to cope with osmotic changes and alterations in the salt ratios presented in DC as well as the capability to tolerate a broader range of DC-salinities with a satisfactory growth (Matos et al., 2016b). This investigation is an extension of previous study on the use of DC as a potential substrate for N. gaditana cultivation (Matos et al., 2015), which advanced our understating of the effect of optimal DC concentration on algal growth. The aims of the present study are: (1) to confirm the optimal DC concentration for N. gaditana growth and subsequently evaluate the effect of DC concentrations on the production of biomass and lipid productivities; (2) to evaluate the performance of N. gaditana under autotrophic, mixotrophic and heterotrophic culture conditions; and (3) to determine the effect of different photoperiod regimes on cell growth, biomass concentration, protein/lipids production and fatty acids composition by exposure to four different light/dark (L/D) cycles, 24L:00D, 16L:08D, 12L:12D, and 08L:16D. 2. Material and methods 2.1. Algal strain and culture The marine species Eustigmatophyceae Nannochloropsis gaditana (clone 130) was kindly supplied by Banco de Microorganismos Marinhos Aidar & Kutner (BMA&K) of the Oceanographic Institute at the University of São Paulo and maintained in autoclaved F/2 seawater medium (Table 1) suitable for marine algae (Guillard, 1975). 2.2. Inland desalination concentrate 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 was determined according to standard methods (APHA, 2005). The Cl, Fe, ammonium (NH+4), total phosphorus (PO3 4 ) and total hardness (CaCO3) values were determined using a Hach Spectrophotometer (Loveland, USA). Total nitrogen (TN) and sulfate were measured photometrically using PermachemÒ Reagent kits. A Shimadzu atomic absorption spectrophotometer (Kyoto, Japan) was used to determine Ca and Mg and a flame photometer (Diadema, Brazil) to determine K and Na. The conductivity and total dissolved solids were measured using a Tecnal conductivity meter (Piracicaba, Brazil) and the pH was measured using a Digimed pHmeter (Campinas, Brazil). The DC is rich in Cl, Na+ and Ca2+ and contains other nutrients (N and P) and trace elements necessary for microalgae growth,

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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Â.P Matos et al. / Bioresource Technology xxx (2016) xxx–xxx Table 1 Compositions of desalination concentrate (DC) and F/2 medium. DC

Value (mean ± SD)

F/2 medium

pH Conductivity (mS cm1)

8.5 ± 0.5 4.5 ± 0.5

Total Dissolved Solids (mg L1) Cl (mg L1) Na (mg L1)

2190.5 ± 156.3

pH 8.0 ± 0.5 Conductivity (mS 35.0 ± 0.5 1 cm ) Stock solution (1.0 mL L1)

CaCO3 (mg L1)

985.2 ± 68.3

SO2 4

(mg L

1

)

1691.3 ± 102 987.5 ± 99.5

138.0 ± 3.2

Ca (mg L1) K (mg L1)

126.5 ± 12.3 47.0 ± 0.2

1 ) NO 3 -N (mg L

30.0 ± 2.2

Mg (mg L

1

)

NH+4 (mg L1) 1 PO3 ) 4 -P (mg L Fe (mg L1)

4.74 ± 0.8 1.35 ± 0.5 0.70 ± 0.1 0.13 ± 0.05

Value

NaNO3 (g L1) 75 NaH2PO4 H2O 5 (g L1) Na2SiO3 9H2O 30 (g L1) Trace metal solution (1.0 mL L1) FeCl3 6H2O (g L1) 3.15 Na2EDTA 2H2O 4.36 (g L1) CuSO4 5H2O 9.8 (g L1) Na2MoO4 2H2O 6.3 (g L1) ZnSO4 7H2O (g L1) 22.0 CoCl2 6H2O (g L1) 10.0 MnCl2 4H2O 180.0 (g L1) Vitamin solution (0.5 mL L1) Thiamine HCl 200 (mg L1) 1.0 Biotin (g L1) Cyanocobalamin 1.0 (g L1)

including Si, K+, Mg+2, Fe+3. The average electrical conductivity of the DC is 4.5 ± 0.5 mS cm1 (Table 1). 2.3. Experimental procedures 2.3.1. Optimal DC concentration for N. gaditana growth The experimental medium contained DC concentrations of 25%, 50%, 75% and 100% in F/2 medium (based on seawater), while F/2 medium with 0% DC was used to conduct control tests. An initial N. gaditana concentration of 105 cells mL1 was used for the preparation of the inoculum. Autotrophic cultures were performed in 500-mL Erlenmeyer flasks with incubation at room temperature (25 ± 2 °C), sparged with atmospheric saturated air-CO2 at concentration of 2–3% vol/vol and a flow rate of 1.5 L min1, using a photoperiod regime of 12L:12D, under a light intensity of 80 lmol photon m2 s1 provided by cool white fluorescent lamps (LUMILUX, OSRAM, Brazil). The incubations were done in duplicate over 7 days of cultivation. When the N. gaditana reached the stationary phase, samples were collected and centrifuged (Nova Técnica, Piracicaba, Brazil) at 4000 rpm for 20 min., washed with ammonium formate (Zhu and Lee, 1997) and centrifuged again. The algal pellet was transferred to a dish and dried in a dehydrator at 45 °C (Zhejiang, China) for further analysis. 2.3.2. Effect of trophic conditions and photoperiod on N. gaditana cultures at optimal DC concentration Experiments were carried out using a in a 4.0 L working volume in a 6.0 L photobioreactor (New Brunswick Bioflo 2000 Fermenter, New Jersey, US) operating in batch mode. The photobioreactor was sterilized by autoclaving at 121 °C for 15 min. prior to the algal cultivation. An F/2 culture medium with DC concentration of 75% and a conductivity of 8200 ± 500 lS cm1, was used in all experiments. Each experiment started with a pre-culture grown at 105 cells mL1 in the exponential phase, which was diluted to 10% and cultivated for 9 days. The airflow in the photobioreactor

was provided via filtered saturated air-CO2. The temperature was kept at 25 ± 1 °C and the culture was continuously agitated (75 rpm). In order to identify the growth medium for N. gaditana which would provide the highest biomass and lipid productivity, three different trophic conditions (autotrophic, mixotrophic and heterotrophic) and four different photoperiod regimes, i.e., light/dark (L/D) cycles of 24L:00D, 16L:08D, 12L:12D, and 08L:16D were studied. For autotrophic cultivation, the cultures were illuminated with a light intensity of 100 lmol photon m2 s1 by four 20 W cool white fluorescent lamps directed to the surface of the bioreactor at different photoperiods. For mixotrophic and heterotrophic cultivations, glucose was used as a carbon source at a concentration of 2.0 g L1, with and without light illumination, respectively. When the N. gaditana cells reached the stationary growth phase, the culture was harvested by centrifugation using a Heinz Janetzki SD600 centrifuge (Engelsdorf-Leipzig, Germany) at 3500 rpm for 25 min., washed with ammonium formate (Zhu and Lee, 1997) and then centrifuged again. The algal pellet was transferred to a porcelain crucible and dried in an oven at 40 °C prior to further analysis. 2.4. Determination of algal growth and biomass Cell counting was performed every day using a hemocytometer coupled to a microscope (Olympus, Germany). The growth rate of each culture was calculated based on the following relationship:

ln

X ¼ lt; X0

ð1Þ

where X0 = initial biomass concentration, at time; X = final biomass concentration and l = specific growth rate of the culture (day1). The biomass concentration was measured, every two days of cultivation, 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 using Eq. (2):

BP ¼ ðX t  X 0 Þ=ðt x  t 0 Þ

ð2Þ 1

where Xt is the biomass production (g L ) at the end of the exponential growth phase (tx) and X0 is the initial biomass production (g L1) at t0 (day1). Cellular protein and lipid productivities were 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) as follows: 1

Lipid productivityðLP ; mg Lip: L1 d Þ ¼ BP  lipid=biomassðw=wÞ

ð3Þ 1

Protein productivityðPP ; mg Prot: L1 d Þ ¼ BP  protein=biomassðw=wÞ

ð4Þ

2.5. Biochemical analysis Total nitrogen was determined by the Kjeldahl method after acid digestion (AOAC 991.20), ammonium addition, steam distillation and titration with 0.1 N HCl (AOAC, 2005). Protein content was calculated using a nitrogen-to-protein conversion factor of N  4.78 (Lourenço et al., 2004). After acid digestion with 4.0 N HCl for 6 h, intracellular lipids were extracted with petroleum ether by the Soxhlet method (AOAC 963.15), concentrated in a rotary evaporator, dried in an oven and weighed (AOAC, 2005). The fatty acids composition was determined after converting the fatty acids to their corresponding fatty acid methyl esters

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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Â.P Matos et al. / Bioresource Technology xxx (2016) xxx–xxx

(FAME), which were determined by gas chromatography using a Shimadzu GC-2014 chromatograph (Kyoto, Japan), equipped with a split-injection port, flame-ionization detector and Restek capillary column (length = 105 m; ID = 0.25 mm) coated with 0.25 lm of 10% cyanopropylphenyl and 90% biscyanopropylsiloxane. Injector and detector temperatures were 260 °C, the oven temperature was initially set at 140 °C for 5 min and, programmed to increase at 2.5 °C min1 to 260 °C, which was held for 30 min. The injection volume was 1 lL, and the split ratio was 10:1. Nitrogen was used as the carrier gas (flow rate 2.2 mL min1) at a constant pressure of 130.3 pKa. FAMEs were identified by comparison with the retention time of individual standards (Sigma, St. Louis, USA). The proportions of each individual acid was calculated from the ratio its peak area and total area for all acids and expressed as a mass percentage. 2.6. Statistical analysis Statistical analysis was performed by one-way analysis of variance (ANOVA) using STATISTICA Software (version 7.0; StatSoft Inc). A p-value of <0.05 was considered to indicate statistical significance, and in cases where significant differences were observed the means for the different treatments were compared pairwise using the Tukey test. 2.6.1. Principal component analysis (PCA) The biomass, lipid/protein contents, main fatty acids (C14:0, C16:0, C18:1, C18:2, C18:3 and C20:5ɷ3) and fatty acids properties (SFA, MUFA and PUFA) were selected for PCA using the Statistica 7.0 software (Statsoft Inc. 2004). All variables were normalized using (n – 1), and Kaiser normalization was performed prior to construction of the PCA biplot. In the resulting PCA biplot, the A, B, C, and D groups were circled according to their distinct cluster formation. 3. Results and discussion 3.1. Effect of DC concentration on biomass and lipid productivities of N. gaditana To assess the applicability of DC as a culture medium, it was mixed with the F/2 medium before the algal cultivation. The compositions of DC and F/2 are shown in Table 1. Different DC concentrations were tested to determine their effect on microalgal growth, measured as biomass and lipid productivities. Productivity results, as well as the conductivity for each DC concentration, are shown in Table 2. Briefly, biomass productivity (BP) in control culture (0% DC) was 101 mg L1 day1, increased slightly at 25% and 50% DC and was maximal (126 mg L1 day1) at 75% DC. At 100% DC the BP decreased to 46 mg L1 day1, likely because of the inability to maintain satisfactory growth at the mineral composition of DC only, indicating that DC needs to supplemented with conventional media. These results are in excellent agreement with

previous data that marine N. gaditana produced more biomass when grown in DC mixed with F/2 medium than in F/2 medium only (Matos et al., 2015). The lipid productivity (LP) showed a comparable trend and increased proportionally with increasing DC concentration from 5.4 mg L1 day1 in control F/2 medium to a maximum of 14.5 mg L1 day1 at 75% DC. At 100% DC the LP decreased close to control levels (5.4 mg lip. L1 day1) (Table 2). Functionally, increased intracellular lipid productivity in N. gaditana at 50–75% DC can be explained by an adaptive osmoregulatory mechanism to cope with either rapid or gradual changes in salinity/conductivity caused by the DC-medium, which are associated with the algal cell membrane permeability (Bartley et al., 2013; Lawton et al., 2015). Our results with marine alga N. gaditana confirm that cells produce more lipids when exposed to hypotonic salinity/conductivity, which was also observed by An et al. (2013) for the lipid content of Chlamydomonas ICE-L exposed to hypotonic conditions. More importantly, these data indicate that the mineral composition of the conventional F/2 medium can be partially replaced by the microelements present in DC to create favorable conditions for microalgal cultivation. For this reason, we conducted the subsequent studies using the optimum DC concentration of 75%. 3.2. Effect of trophic conditions and photoperiod regimes on algal growth and biomass The aim of this experiment was to evaluate the effects of different trophic conditions and light/dark cycles on culturing N. gaditana in a medium with the optimum DC concentration of 75% by measuring growth rate and biomass productivity. Several microalgal species have the ability to switch between phototrophic and heterotrophic metabolisms. Heterotrophic growth eliminates the need for illumination, which is required for cultivation under autotrophic conditions. However, light has multiple effects on photosynthetic organisms: it provides not only energy to support metabolism, but is also a fundamental signal that influences many intracellular components (Simionato et al., 2011). The alga N. gaditana was grown in batch cultures exposed to four different light/dark cycles (24L:00D, 16L:08D, 12L:12D, 08L:16D) under three trophic conditions: autotrophic, mixotrophic and heterotrophic. For all cultures the growth kinetics was monitored through the increase in cell number. Fig. 1A shows that growth rates differ significant for most trophic conditions and light/dark cycles. Autotrophic conditions coupled with a 16L:08D cycle resulted in a maximum cell density (MCD) of 2.9  107 cells mL1, while mixotrophic and heterotrophic conditions resulted in cell densities of 1.3  107 cells mL1 and 6.6  106 cells mL1, respectively. N. gaditana cultures grew fastest under autotrophic culture conditions (l ranging from 0.40 to 0.62 day1), followed by mixotrophic (l ranging from 0.43 to 0.56 day1), and heterotrophic (0.38 day1) conditions (Fig. 1B). Biomass concentrations differ significantly (p < 0.05) under the three trophic conditions, and were maximal at mixotrophic (0.60 to 1.25 g L1),

Table 2 Effect of desalination concentrate (DC) concentrations on biomass productivity (BP) and lipid productivity (LP) of marine Nannochloropsis gaditana.

*

Cultivation

BP (mg L1 day1)

LP (mg L1 day1)

Conductivity (mS cm1) Initial

Final

0% DC* 25% DC 50% DC 75% DC 100% DC

101 ± 9ab 90 ± 8c 115 ± 4b 126 ± 8a 46 ± 3d

5.0 ± 2.6c 5.4 ± 2.1c 10.5 ± 2.2b 14.5 ± 5.1a 5.4 ± 2.1c

35.1 ± 0.4 15.5 ± 1.3 10.2 ± 1.0 8.0 ± 1.2 4.5 ± 1.1

38.2 ± 0.5 16.8 ± 0.6 12.8 ± 1.0 10.2 ± 0.8 5.6 ± 0.4

Control test (F/2 medium based on seawater). Different letters in the same column correspond to significant differences (p < 0.05).

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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Moreover, N. gaditana cells grown under continuous illumination (24L:00D) produce less lipids (LP, 5.8–9.9 mg lip. L1 day1) in comparison with cultures exposed to light/dark cycles of 16L:08D, 12L:12D and 08L:16D, with LP ranging from 13.7– 15.3 mg lip. L1 day1 (Table 4). Comparable LP values of 8.5– 10.0 mg lip. L1 day1 were reported for euryhaline N. gaditana cells exposed to photoperiods of 24L:00D, 16L:08D, 12L:12D, and 08L:16D Mitra et al. (2015). In summary, a maximum BP value was observed for marine N. gaditana cultured using a DC concentration of 75%, under mixotrophic conditions and a light/dark cycle of 16L:08D, which is thus the most appropriate treatment for this species.

10000

Cell density (mL-1 x104)

A 1000

100 Aut. 24:0 h

Aut. 16:8 h

Aut. 12:12 h

Aut. 8:16 h

Mix. 24:0 h

Mix. 16:8 h

Mix. 12:12 h

Mix. 8:16 h

Het. (dark)

10

0

Duplication rate (days-1)

0,8

2

4 6 Cultivation time (days)

8

10

B 3.3. Effect of experimental conditions on protein and lipid productivities

0,6

0,4

0,2

0,0

Trophic and light variation

Fig. 1. Nannochloropsis gaditana growth under different trophic conditions and light regimes of 24L:00D, 16L:08D, 12L:12D and 08L:16D. (A) Increase in cell number according to cultivation time (days), (B) Specific growth rate during exponential phase (l) calculated for all growth curves. All data were obtained by merging the results of at least two independent experiments.

intermediate at heterotrophic (0.85 g L1) and minimal at autotrophic (0.30 to 0.77 g L1) conditions (Table 3). In addition, N. gaditana showed a maximum BP of 142 mg L1 day1 under mixotrophic conditions and a 16L:08D cycle. Higher cell densities under mixotrophic conditions could be due to higher amounts of available energy contributing to aerobic respiration coupled with catabolism of the glucose present in the medium along with photosynthesis (Mitra et al., 2012). The synergetic effect between light and organic substrates in terms of algal growth has been reported before (Cheirsilp and Torpee, 2012). Under complete darkness (i.e., heterotrophic condition), N. gaditana cells produced intermediate amounts of biomass (BP = 85 mg L1 day1), while cultures exposed to continuous illumination (24L:00D) reached the stationary phase earlier with a lower BP (29 mg L1 day1), indicating photo-inhibition caused by excess illumination (Table 3).

This experiment examined the effect of different experimental conditions of the intracellular compositions, i.e. protein and lipid productivities of marine N. gaditana. Using a nitrogen-to-protein (N-to-P) conversion factor of N  4.78 (Lourenço et al., 2004), protein contents varied from 17.9 to 44.8% (Table 4), with statistical different protein contents obtained for the different treatments (p < 0.05). The protein content was highest for the mixotrophic culture conditions, with an average of 30.1% (range 17.9–44.8), followed by autotrophic, with an average of 23.4% (range 20.2– 27.5%), and heterotrophic (23.2%) condition, the latter two average values being statistically equal (p < 0.05). These results are comparable to data, for instance by Tibbetts et al. (2015), who found a protein content of 35.0% for N. granulata, using the same conversion factor of N  4.78. With regard to the algal lipids, total lipid contents vary from 10.5 to 16.7%, which in some cases translated to a relatively high lipid productivity when coupled to high biomass productivity. Interestingly, N. gaditana has a remarkable ability to accumulate protein and lipids under quite different culture conditions. For example, N. gaditana cultures show optimal protein synthesis (37.3 to 44.8%) under mixotrophic conditions and a photoperiod of 16L:08D or 12L:12D. In contrast, comparable lipid accumulation occurred in N. gaditana cells under autotrophic conditions and a 16L:08D cycle (16.7%) and under mixotrophic conditions and a 08L:16D cycle (15.7%). Under heterotrophic condition, a lipid content of 12.1% was noted (Table 4). It appears that under autotrophic conditions, N. gaditana requires a longer light period, i.e., a 16L:08D cycle for a high lipid productivity (15.9 mg L1 day1). With a shorter light period as in a 08L:16D cycle, high lipid productivity (15.3 mg L1 day1) requires mixotrophic cultivation (Fig. 2). According to Mitra et al. (2012), shorter light periods during mixotrophic cultivation may act as a stress signal, prompting the cells

Table 3 Growth parameters of N. gaditana cultured with optimal DC concentration of 75% under different experimental conditions. Data are expressed as means ± SD (n = 3). Maximum cell density (cell mL1  104)

Specific growth rate (day1)

Biomass concentration (g L1)

Biomass productivity (BP, mg L1 day1)

Autotrophic (light/dark) 24L:00D 16L:08D 12L:12D 08L:16D

400 ± 9a 2900 ± 14d 1200 ± 20b 1180 ± 15b

0.50 0.62 0.50 0.40

0.30 ± 0.10a 0.45 ± 0.05ab 0.77 ± 0.10abc 0.35 ± 0.05a

28 ± 4a 42 ± 6abc 88 ± 10cd 35 ± 7ab

Mixotrophic (light/dark) 24L:00D 16L:08D 12L:12D 08L:16D Heterotrophic (dark)

1020 ± 17b 1700 ± 11c 1654 ± 16c 1010 ± 13b 665 ± 10ab

0.43 0.45 0.53 0.56 0.38

0.60 ± 0.15ab 1.25 ± 0.10c 0.98 ± 0.14bc 0.82 ± 0.12abc 0.85 ± 0.10abc

30 ± 7a 142 ± 12e 112 ± 8e 80 ± 8bcd 85 ± 13cd

Values in the same column with different superscript letters are significantly different (p < 0.05).

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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Table 4 Protein and lipid productivities of N. gaditana cultured with optimal DC concentration of 75% under different experimental conditions. Data are expressed as means ± SD (n = 2). Protein (%)

Lipid (%)

Protein prod. (PP, mg prot. L1 day1)

Lipid prod. (LP, mg lip. L1 day1)

Autotrophic (light/dark) 24L:00D 16L:08D 12L:12D 08L:16D

20.2 ± 0.2ab 23.5 ± 0.3b 27.5 ± 0.5c 22.5 ± 0.4b

10.7 ± 0.6a 16.7 ± 0.5c 12.0 ± 0.8ab 10.5 ± 1.0a

18.8 ± 1.2bc 21.9 ± 0.8bc 31.4 ± 2.3cd 22.5 ± 0.7b

9.9 ± 0.5b 15.9 ± 0.7e 13.7 ± 0.7d 10.5 ± 0.2b

Mixotrophic (light/dark) 24L:00D 16L:08D 12L:12D 08L:16D Heterotrophic (dark)

20.5 ± 0.9ab 37.3 ± 0.3d 44.8 ± 1.3e 17.9 ± 0.1a 23.2 ± 0.2b

11.6 ± 1.2ab 11.8 ± 0.8ab 11.3 ± 0.7a 15.7 ± 0.3bc 12.1 ± 0.4ab

10.2 ± 0.8a 42.3 ± 1.6d 51.2 ± 2.1e 17.4 ± 0.9ab 23.2 ± 1.0bc

5.8 ± 0.1a 12.8 ± 0.5c 12.9 ± 0.3c 15.3 ± 0.6e 12.1 ± 0.4bc

Values in the same column with different superscript letters are significantly different (p < 0.05).

Step 2a

N. gaditana (F/2 medium) LP = 5.0 mg L-1 day-1

Step 1

N. gaditana (75% DC medium) Autotrophic/16L:08D LP = 15.9 mg L-1 day-1

N. gaditana (75% DC medium) LP = 14.5 mg L-1 day-1 Step 2b

N. gaditana (75% DC medium) Mixotrophic/08L:16LD LP = 15.3 mg L-1 day-1

Fig. 2. Summary scheme showing how to enhance lipid productivity (LP, mg L1 day1) in marine N. gaditana through the manipulation of growth parameters. Step 1 = optimization of culture medium based on desalination concentrate (DC) which affects alga through the change in salinity/conductivity; Step 2a = enhancing LP of alga after establishing the optimal photoperiod under autotrophic culture conditions; Step 2b = enhancing LP of alga after adding an extra carbon source (glucose, 2.0 g L1) with optimal photoperiod under mixotrophic culture conditions.

to convert the excess glucose that is available in the medium into storage lipids and minimize cell division. This stress-adaptation phenomenon is similar to changes in response to salinity or nitrogen limitation and increased lipid production in other algae (Matos et al., 2014; Selvaratnam et al., 2015). Simionato et al. (2011) found that nutrient starvation during the stationary phase is the major factor that induces lipid accumulation in N. gaditana cells during acclimation to different illumination regimes, and that the light intensity per se does not influence lipid accumulation. Nevertheless, the illumination conditions can play an indirect but important role when combined with other factors that influence photosynthesis and growth, such as salinity and nutrient supplementation or depletion. In this study, we hypothesized that algae cultured autotrophically (with only air-saturated CO2 as an inorganic carbon supply), accumulate lipids to counterbalance the gradual changes in salinity/conductivity triggered by the DC-medium, representing a ‘stress condition’. Furthermore, algae probably accumulate lipids under mixotrophic cultivation, due to their ability to assimilate glucose as an extra carbon source, suggesting that increased availability of carbon leads to an increased proportion of stored lipids. The light/dark cycle was found, in this study, to have a crucial impact on biomass productivity of N. gaditana, which is directly correlated with lipid productivity, indicating that the illumination photoperiod is an important factor that need to be taken into consideration. 3.4. Effect of trophic conditions and photoperiod on fatty acids composition The fatty acids (FAs) composition of marine N. gaditana cultured in DC at the optimum concentration is shown in Table 5. Under all test conditions the predominant FAs in marine N. gaditana, are (expressed as percentage of total FAs) palmitic acid (C16:0; 25.1– 54.5%) and margaric acid (C17:0; 4.4–28.6%), representing 56.0% of the total FA content. FAs present at intermediate levels are oleic

acid (C18:1; 2.2–12.3%), linoleic acid (C18:2; 3.7–22.7%) and linolenic (C18:3; 0.5–13.3%), representing 27.3% of the total FA contents. The FAs myristic acid (C14:0; 0.8–12.0%) and eicosapentaenoic acid (C20:5ɷ3; 0.1–5.6%) were present at trace levels and represent 9.2% of the total FA content. In general, autotrophic, heterotrophic and mixotrophic cultivation conditions the main FAs are saturated fatty acids (SFAs; 33.8–84.7%), polyunsaturated fatty acids (PUFAs; 10.2–46.0%) and monounsaturated fatty acids (MUFAs; 5.0–24.1%). As shown in Table 5, N. gaditana cells that were cultured autotrophically tend to produce higher levels of SFAs (mainly C14:0 and C16:0) at an average of 70.0% of total FAs, especially when a 16L:08D cycle photoperiod was applied (SFA 85.0% of total FAs), consistent with previous data on this saturated fatty acid (Matos et al., 2015). On the other hand, when adding glucose to the optimal DC medium, more PUFAs are formed. For example, in cultures grown in complete darkness (i.e. heterotrophic condition), N. gaditana synthesized PUFAs at high concentrations (46.0% of the total FAs), which were mainly linoleic (C18:2, 26.6% of total FAs), linolenic (C18:3, 13.8% of total FAs) and eicosapentaenoic (C20:5ɷ3, 5.6% of total FAs) acids. The cultures grown under mixotrophic culture conditions resulted in the production of a balanced ratio of saturated (SFA) and unsaturated (MUFA + PUFA) fatty acids. Overall, for the marine N. gaditana cells cultured in optimal DC-medium, the microalga was virtually devoid of long-chain fatty acids (e.g. C20, and C22) and showed a trend toward an increased formation of medium-chain (C16 and C18) fatty acid methyl esters, which is an ideal lipid source for biodiesel production (Cheirsilp and Torpee, 2012; Ji et al., 2014). According to Fernandes et al. (2016), SFAs and MUFAs in microalgae are produced by de novo FA synthesis in the chloroplast and provide the substrates needed for PUFA biosynthesis in the endoplasmic reticulum. In addition, algal diets with low (SFA + MUFA)/PUFA ratios (i.e. <2.00) are optimal for the feeding of larval and juvenile oysters. Our data indicate that the (SFA + MUFA/

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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Table 5 Fatty acids composition (% of total fatty acid content) of N. gaditana cultured with optimal DC concentration of 75% under different experimental conditions. Data are expressed as means ± SD (n = 2). C16:0

C17:0

C18:1

C18:2

C18:3

C20:5ɷ3

SFA

MUFA

PUFA

(SFA + MUFA)/PUFA

Autotrophic (light/dark) 24L:00D 1.3 ± 0.1 16L:08D 10.5 ± 0.5 12L:12D 1.4 ± 0.2 08L:16D 7.2 ± 0.7

C14:0

33.5 ± 0.7 42.9 ± 1.6 54.5 ± 1.8 50.5 ± 1.5

2.4 ± 0.3 28.6 ± 1.2 9.5 ± 0.8 20.1 ± 1.3

12.3 ± 0.7 9.1 ± 0.6 9.7 ± 0.8 2.2 ± 0.1

12.9 ± 0.9 4.3 ± 0.2 3.7 ± 0.3 11.8 ± 0.7

2.5 ± 0.1 0.7 ± 0.1 1.5 ± 0.2 2.3 ± 0.3

0.7 ± 0.1 1.7 ± 0.2 0.3 ± 0.1 0.1 ± 0.1

46.5 ± 2.5 84.7 ± 3.6 69.5 ± 2.7 79.4 ± 2.2

24.1 ± 1.6 5.0 ± 0.9 14.5 ± 1.1 5.8 ± 0.7

17.3 ± 0.8 10.2 ± 0.7 15.9 ± 1.1 14.7 ± 1.2

4.08 8.79 5.28 5.79

Mixotrophic (light/dark) 24L:00D 8.8 ± 0.6 16L:08D 8.8 ± 0.4 12L:12D 12.0 ± 1.0 08L:16D 7.2 ± 0.4 Heterotrophic (dark) 0.8 ± 0.1

35.1 ± 1.1 36.0 ± 1.3 25.1 ± 1.3 39.0 ± 1.2 25.5 ± 1.2

15.3 ± 0.7 26.0 ± 1.4 4.6 ± 0.2 18.4 ± 0.9 5.8 ± 0.4

4.9 ± 0.4 2.8 ± 0.2 5.1 ± 0.3 5.5 ± 0.3 12.2 ± 0.3

22.7 ± 0.4 9.6 ± 0.6 19.2 ± 0.7 16.8 ± 0.7 26.6 ± 0.7

2.0 ± 0.2 0.5 ± 0.1 0.3 ± 0.4 1.8 ± 0.1 13.8 ± 0.8

1.4 ± 0.1 3.3 ± 0.2 0.5 ± 0.1 0.2 ± 0.1 5.6 ± 0.1

62.0 ± 2.0 67.5 ± 2.4 46.3 ± 2.5 67.3 ± 2.3 33.8 ± 1.0

11.2 ± 0.9 10.3 ± 0.7 11.2 ± 1.1 11.9 ± 1.1 20.1 ± 0.7

26.7 ± 0.7 14.3 ± 0.7 20.0 ± 1.4 20.8 ± 0.8 46.0 ± 1.4

2.74 5.54 2.80 3.80 1.17

Note: SFAs not shown in the table: Tridecanoic acid, C13:0; Myristic acid, C14:0; Pentadecanoic acid, C15:0; Behenic acid, C22:0. MUFAs not shown in the table: Margaroleic acid, C17:1; Erucic acid, C22:1; Nervonic acid, C24:1. PUFAs not shown in the table: Eicosatrienoic acid, C20:3; Arachidonic acid, C20:4; Docosahexaenoic acid, C22:6.

PUFA) ratio decreases in the order of autotrophic (6.15) > mixotrophic (3.35) > heterotrophic (1.15) culture conditions (Table 5), suggesting that heterotrophic cultivation is most suitable to produce this microalga for aquaculture. Although the lipid profiles of the marine N. gaditana biomass varied, the microalga mainly accumulated the following fatty acids (C): 14:0; 16:0; 17:0; 18:1; 18:2; 18:3 and 20:5. Therefore, the microalga study herein is industrially important for pharmaceutical and nutritional supplements for humans and animal feed (polyunsaturated fatty acids), and the production of biofuels, mainly biodiesel (Mitra et al., 2015; Tibbetts et al., 2015; Varshney et al., 2015). 3.5. Principal component analysis Given the large number of variables, the amounts of biomass, proteins, lipids and main fatty acids produced under different trophic and photoperiod regimes were selected for principal component analysis (PCA). The PCA biplot (Fig. 3) revealed four important clusters based on the PC 1 and PC 2 with variabilities of 44.86% and 18.52%, respectively. Cluster A was encircled with the variables C18:2, C18:3, C20:5ɷ3, PUFA and heterotrophic culture condition,

while cluster B consisted of variables C18:1 and MUFA. Cluster C was encircled of the variables photoperiod (12L:12D, 16L:08D and 24L:00D), biomass, protein and mixotrophic culture conditions. Cluster D was encircled with variables photoperiod (08L:16D, 12L:12D, 16L:08D), lipids, C14:0, C16:0, SFAs and autotrophic culture conditions. The autotrophic culture condition with 16L:08D photoperiod in cluster D were positively associated with lipids and SFAs along with C14:0 and C16:0 (Fig. 3), indicating that a 16L:08D photoperiod is optimal for the maximal production of SFAs. In addition, higher SFA production is caused mainly due to the synthesis of dominant C16:0 that contributes to the highest SFA + MUFA)/PUFA index of 8.79 at this photoperiod regime (Table 5). Interestingly, at the same 16L:08D photoperiod PUFA content was the lowest (10.2% of total FAs) of all treatments and is thus negatively associated with PUFA. It should be noted that under mixotrophic culture conditions the 16L:08D and 12L:12D cycles increased the protein levels (cluster C), whereas the C18:1 and MUFA were lonely located in the cluster B. with regard to variables that are located in cluster A, the unsaturated fatty acids (i.e. C18:2, C18:3, C20:5ɷ3) and PUFA

Fig. 3. Principal component analysis (PCA) of the biomass, protein, lipid and main fatty acids composition under variable trophic and light photoperiod conditions, based on a culture medium with optimal desalination concentrate (DC) of 75%.

Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004

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were strongly correlated with heterotrophic culture condition, indicating that N. gaditana cells have a tendency to synthesize more PUFA in complete darkness. The PCA biplot clearly demonstrates that the 16L:08D cycle during autotrophic cultivation is the best condition for optimal SFA production, which is an important finding, since RSFA and RMUFA are very important for biodiesel production (Cheirsilp and Torpee, 2012). 4. Conclusions Nannochloropsis gaditana was shown to be capable of higher lipid production in culture medium with optimal DC concentration compared to control medium. By varying the growth conditions (trophic mode, photoperiod and salinity based on DC conductivity) the chemical composition (protein, lipids and fatty acids) of N. gaditana can be manipulated. Under autotrophic conditions, the algae synthesize more SFAs, while by adding an addtional carbon source to the DC medium, the percentage of PUFAs is increased. The present study has identified an effective strategy for the optimization of culture medium based on DC for enhancing algal lipid contents. Acknowledgements This work was supported by FAPEU (Fundação de Amparo à Pesquisa e Extensão Universitária) under project number 213/1999. AP Matos is grateful to CAPES – Brazil for providing a doctoral scholarship. We are also grateful for the laboratory facilities provided by the Laboratory of Analysis (LABCAL/UFSC) for this research. References Abad, S., Turon, X., 2012. Valorization of biodiesel derived glycerol as a carbon source to obtain added-value metabolites: focus on polyunsaturated fatty acids. Biotechnol. Adv. 30, 733–741. An, M., Mou, S., Zhang, X., Zheng, Z., Ye, N., Wang, D., Zhang, W., Miao, J., 2013. Expression of fatty acid desaturase genes and fatty acid accumulation in Chlamydomonas sp. ICE-L under salt stress. Bioresour. Technol. 149, 77–83. AOAC, 2005. AOAC official method 963.15, 991.20. In: Official Methods of Analysis of AOAC International. 18th ed. AOAC International, Gaithersburg. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Bartley, M.L., Boeing, W.J., Corcoran, A.A., Holguin, F.O., Schaub, T., 2013. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina invading organisms. Biomass Bioenergy 54, 83–88. Carioca, J.O.B., Hiluy Filho, J.J., Leal, M.R.L.V., Macambinra, F.S., 2009. A hard choice for alternative biofuels to diesel in Brazil. Biotechnol. Adv. 27, 1043–1050. Cheirsilp, B., Torpee, S., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour. Technol. 110, 510–516. Dickinson, K.E., Whitney, C.G., McGinn, P.J., 2013. Nutrient remediation rates in municipal wastewater and their effect on biochemical composition of the microalga Scenedesmus sp. AMDD. Algal Res. 2, 127–134. Fernandes, T., Fernades, I., Andrade, C.A.P., Cordeiro, N., 2016. Changes in fatty acid biosynthesis in marine microalgae as response to medium nutrient availability. Algal Res. 18, 314–320. Guillard, R.R.L., 1975. Culture of phytoplankton for feeding marine invertebrates. In: Smith, W.L., Charley, M.H. (Eds.), Culture of marine invertebrate animals. Plenum, New York, pp. 29–60. Henkanatte-Gedera, S.M., Selvaratnam, T., Caskan, N., Nirmalakhandan, N., Voorhies, W.V., Lammers, P.J., 2015. Algal-based, single-step treatment of urban wastewater. Bioresour. Technol. 189, 273–278. Isleten-Hosoglu, M., Gultepe, I., Elibol, M., 2012. Optimization of carbon and nutrient sources for biomass and lipid production by Chlorella saccharophila under heterotrophic conditions and development of Nile red fluorescence based

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Please cite this article in press as: Matos, Â.P., et al. Effects of different photoperiod and trophic conditions on biomass, protein and lipid production by the marine alga Nannochloropsis gaditana at optimal concentration of desalination concentrate. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j. biortech.2016.11.004