Protocol for maximizing the triglycerides-enriched lipids production from Dunaliella salina SA32007 biomass, isolated from the Salar de Atacama (Northern Chile)

Diana Arias-Forero, Gladys Hayashida, Mario Aranda, Solange Araya, Tamara Portilla, Andreína García, Paula Díaz-Palma

Advanced Mining Technology Center (AMTC), Faculty of Physic and Mathematics Sciences, Universidad de Chile, Santiago, Chile.
Department of Chemistry, Faculty of Sciences, Universidad Católica del Norte, Antofagasta, Chile.
Laboratory of Advance Research on Foods and Drugs, Department of Food Science, Nutrition and Dietetics, Faculty of Pharmacy, Universidad de Concepción, Concepción, Chile.

DOI: 10.4236/abb.2013.48110                

Abstract

This paper reports the effects of different culture conditions for Dunaliella salina SA32007 from Salar de Atacama (second Region, northern ofChile) over biomass, lipid production and triglycerides synthesis. A maximum value of microalgae density (8.2 × 109 Cells/L) and an intrinsic growth rate (0.17 d-1), were obtained using a culture with 0.5 mol/L of NaCl and a nitrogen/phosphorous (N/P) limitation of 14/1. The triglycerides production was significantly favoured under nitrogen deficiency conditions (Mann-Whitney test; p = 0.0043). However there was a nitrogen-limiting threshold for the stimulation and accumulation of triglycerides (N/P: 14/1), lower than that limit, these compounds would not be accumulated. It was also observed that triglyceride content was not proportional to the total lipid content and the maximum number of cells. The aeration system employed stimulated the growth and synthesis of structural organic molecules. Regarding microalgae growth stage subjected to nitrogen deficiency, when the deficit was applied before the lag phase, the negative effect on the biomass and the triglycerides production decreased.

Keywords

Lipids; Triacilglycerides; Nitrogen Deficiency; Salinity; Dunaliella salina

1. INTRODUCTION

There is now a worldwide crisis due to the need of new sources of energy. Various disciplines are looking for new conventional and renewable materials to generate heat energy [1]. Among these, the use of microalgae biomass to produce oil and biodiesel is considered increasingly relevant [2-4]. Biodiesel generated from algae offers many advantages, e.g. during algae photosynthesis the atmospheric CO₂ is fixed at a similar rate to that produced during combustion process of biodiesel [5,6], thus, the balance of CO₂ pollution generated is virtually zero. In addition, microalgae show a high productivity rate with yields per acre nearly 30 times greater than those of corn or soybeans [7]. However, the oil quality for biodiesel production is not always appropriate because it has low triglycerides content, and high concentration of free fatty acids. The latter can interfere with the trans-esterification process required for the biofuel obtaining [4]. Different culture tests including high salinity and nutrient limitation (e.g. Mg, K, Na, NO₃ and PO₄) can enhance lipid synthesis and particularly triglycerides, but unfortunately these conditions produced a microalgae biomass reduction and therefore, the biodiesel yield also decreases [8-16]. The objective of this study was the assay of specific conditions in salinity and nitrogen deficiency and air bubbling that could stimulate the triglycerides production, while the negative effect over the biomass is mitigated. This will be carried out by a strain of Dunaliella salina SA32007, isolated from the Salar de Atacama, II Region—Chile, in order to potentiate biotechnological properties of the local microorganisms.

Dunaliella salina is a cosmopolitan species living in hypersaline aquatic environments [17]. In Chile there are records of Dunaliella salina in saline lagoons located in Highland Andean Salars, as in Salar de Atacama (Northern, Chile) [18]. High sunlight intensities stimulate this microalgae to produce a variety of carotenoids, being the most abundant in ß-carotene. This pigment is formed as a protective barrier against oxidation due to free radicals caused by sunlight [19]. It is noteworthy that Dunaliella species are recognized primarily for its ability to produce ß-carotene, which is verified by reviewing the vast scientific literature related to this topic [20-23]. Other potentialities of Dunaliella, such as their ability to induce lipids bioaccumulation in large quantities, have also been studied [24].

2. MATERIAL AND METHODS

2.1. Dunaliella salina SA32007 and Media

D. salina SA32007 strain, isolated from the Salar de Atacama (23˚30'0"S, 68˚15'0"W) II Region Antofagasta, Chile, was cultured in two modified versions of Johnson medium [25], one in which the NaCl concentration changed to vary the salinity (identified from here and beyond as “Medium A”) and another that was modified in its N/P ratio (identified from here and beyond as “Medium B”).

“Medium A” contained the following (per liter): NaCl 11.68 g (0.2 M)/29.2 g (0.5 M)/58.4 g (1 M)/116.9 g (2 M)/175.2 g (3 M)/233.6 g (4 M) y 292 g (5 M); MgCl₂·H₂O 1.5 g; MgSO₄·7H₂O 0.5 g; KNO₃ 1 g; KCl 0.2 g; CaCl₂ 0.2 g; KH₂PO₄ 0.071 g (14/1 N/P Ration, Johnson Medium reference [25]); Tris (hydroxymethyl) aminomethane 2.45 g; EDTA·2Na 1.89 mg; ZnSO₄·7H₂O 0.087 g; H₃BO₃ 0.61 mg; CoCl·6H₂O 0.015 mg, CuSO₄·5H₂O 0.06 mg; MnCl₂ 0.23 g, (NH₄)₆Mo₇O₂₄·4H₂O 0.38 mg; Fe(III)·EDTA 3.64 mg; vitamin B1 0.1 mg; vitamin B12 0.5 mg; biotin 0.5 mg, pH was fixed to 8.0 with 1 N HCl. Additionally, in this step was tested the 25/1 N/P ration (KH₂PO₄ 0.04 g), corresponding to these determined in the collection strain area.

“Medium B” contained the following (per liter): KH₂PO₄ 2 g/0.2 g/0.071 g/0.04 g and KNO₃ 1 g (which resulted in a change in the N/P ration equivalent to 0.5/1, 5/1, 14/1, 25/1, respectively); MgCl₂·H₂O 1.5 g; MgSO₄·7H₂O 0.5 g; NaCl 29.2 g; KCl 0.2 g; CaCl₂ 0.2 g; Tris (hydroxymethyl) aminomethane 2.45 g; EDTA·2Na 1.89 mg; ZnSO₄·7H₂O 0.087 g; H₃BO₃ 0.61 mg; CoCl·6H₂O 0.015 mg, CuSO₄·5H₂O 0.06 mg; MnCl₂ 0.23 g, (NH₄)₆Mo₇O₂₄·4H₂O 0.38 mg; Fe(III)·EDTA 3.64 mg; vitamin B1 0.1 mg; vitamin B12 0.5 mg; biotin 0.5 mg, pH was fixed to 8.0 with 1 N HCl. The N/P rations employed in the “Medium B” were modified from the original recipe of Johnson Medium [25], which presents a N/P ratio of 14/1 corresponding to KH₂PO₄ 0.071 g, and KNO₃ 1 g.

Each culture medium was supplemented with chloramphenicol 1%, in order to obtain axenic monocultures.

Experiments were conducted under controlled light and temperature conditions at 30˚C with an incident white light of 360 μmol/m²/s and a photoperiod cycle of 15/9 (light/dark). A high light intensity was selected to maintain the optimal conditions for microalgae acclimated to large amounts of light. In fact, measurements of the light incident on the Salar de Atacama often exceed 500 μmol/m²/s [26].

2.2. Experimental I: Variation of Salt Concentration of NaCl

A preliminary screening of salinity-tolerance was performed because the microalgae strain was kept under non-hipersaline culture conditions for more than three years. Fourteen flasks (E-I1-E-I14) with 200 mL of “Medium A” were inoculated with 10 mL of axenic culture of D. salina SA32007 (average cell density of 8.0 × 10⁶ cells/L) and incubated at 30˚C for 30 days. Salinity range varied between 0.2 M and 5.0 M NaCl as described in Dunaliella salina SA32007 and Media section. Control conditions were set according to the isolation place of the microalgae strain, corresponding to a N/P ratio of 25/1 and a salinity level of 0.5 mol/L of NaCl (Table 1).

2.3. Experimental II: Variation of N/P Ratio, Air Bubbling and Nitrogen Deficiency

In this section different N/P ratios were assayed (Table 2) keeping fixed the salinity at 0.5 mol/L NaCl, which yielded the maximum biomass value in the experimental I. Additionally, two other variables were included in this step: the air supply using bubbling and the point within the growth curve of the strain where the nitrogen deficiency was applied. In the second case, two treatments were tested: one in which the deficit of nitrogen was applied Before the Latency Phase (BLP-treatments) and another in which the deficit was applied after the latency Phase (ALP-treatments). As regards the air bubbling, treatments with bubbling (Bb-treatments) and without

Table 1. Experimental I: Provided by varying the salt concentration of NaCl. Experimental II: Provide by varying the N/P ratio, the bubbling and the time of application of nitrogen deficiency.

^*Control conditions according to the isolation place of the D. salina SA32007.

Table 2. Experimental II: Provide by varying the N/P ratio, the bubbling and the time of application of nitrogen deficiency.

Abbreviators: Bubbled (Bb); Not Bubbled (NBb); Before Lag Phase (BLP); After Lag Phase (ALP). ^*Control conditions according to the isolation place of the D. salina SA32007, varying the air bubbling.

bubbling (NBb-treatments) were tested. These treatments were numerated from E-II3 to E-II14 (Table 2). The initial density of each culture averaged 1147 ± 946 Cells/L. The controls (E-II1 and E-II2) were cultured using the same conditions found at the isolation site of the strain, but varying the bubbling (Table 2).

2.4. Biomass Quantification

Microalgal biomass was daily count in duplicate using Neubauer chambers. Microalgae samples were pre-fixed with a 5% Lugol solution. The maximum number of cells in each culture, measured as the carrying capacity (K), was correlated to its intrinsic growth rate (μ).

2.5. Extraction and Quantification of Total Extractable Lipids and Triglycerides

Lipids from the microalgae pellet obtained after biomass centrifugation, were extracted using the Bligh and Dyer method [27]. Thus, the results represent the Percentage of total extractable lipids (%TELs) from fresh biomass of the microalgae. The Percentage of Triglycerides (%TAGs) in total extractable lipids was determined by the glycerol-3-phosphate-oxidase-p-chlorophenol enzymatic method (Triglyceride GOP/PAP AA from Wiener) using triolein as standard [10,28].

Each experimental result was obtained through a triplicated set of data. Standard deviation was calculated in each case. Additionally, the overall effect of different variable tested in this study was statistically validated through the non-parametric significance test of MannWhitney U [29].

3. RESULTS

3.1. Microalgae Growth Curve and Salinity Assays

Figures 1 (a) and (b) show the growth curves of D. salina SA32007 in Media A and B, respectively. Both cultures (N/P ratio of 14/1 and 25/1) showed that microalgae samples cultured with NaCl levels from 0.2 mol/L to 1 mol/L maintained a lag phase for a period between 7 and 11 days, followed by the corresponding exponential phase. For treatments with salinities higher than 1 mol/L of NaCl (E-I3-E-I7 and E-I10-E-I14) no culture growth was appreciated and the mean cell density values of the initial inoculum remained unchanged; this was observed regardless of their N/P ratio. The kinetic behaviour of each treatment is summarized in Table 3. The maximum growth was achieved using 0.5 mol/L of NaCl and a N/P ratio of 14/1 and reached a value of 8.2 × 10⁹ Cells/L (Table 3).

3.2. N/P Ratio, Air Bubbling and Nitrogen Deficiency

Figure 2(a) indicates the growth curves for microalgae cultures subjected to nitrogen deficiency, applied before and after the lag phase with air bubbling. It was observed

Conflicts of Interest

The authors declare no conflicts of interest.

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