Scholarly article on topic 'Effect of nitrate on lipid production by T. suecica, M. contortum, and C. minutissima'

Effect of nitrate on lipid production by T. suecica, M. contortum, and C. minutissima Academic research paper on "Biological sciences"

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Academic research paper on topic "Effect of nitrate on lipid production by T. suecica, M. contortum, and C. minutissima"

VERSITA

Eur J. Biol. • 8(6) • 2013 • 578-590 DOI: 10.2478/s11535-013-0173-6

Central European Journal of Biology

Effect of nitrate on lipid production by T suecica, M. contortum, and C. minutissima

Research Article

Didier Sánchez-García1, Anayelli Resendiz-Isidro1, Thelma Lilia Villegas-Garrido2, César Mateo Flores-Ortiz3, Benjamín Chávez-Gómez4, Eliseo Cristiani-Urbina1*

1Department of Biochemical Engineering, National School of Biological Sciences, National Polytechnic Institute, 11340 Mexico DF, Mexico

2Department of Biophysics, National School of Biological Sciences, National Polytechnic Institute, 11340 Mexico DF, Mexico

3Biotechnology and Prototypes Unit, Faculty of Higher Studies-Iztacala, National Autonomous University of Mexico, 54090 State of Mexico, Mexico

4Mexican Petroleum Institute, 07730 Mexico DF, Mexico

Received 28 August 2012; Accepted 23 February 2013

Abstract: Microalgae are an alternative and sustainable source of lipids that can be used as a feedstock for biodiesel production. Nitrate is a good nitrogen source for many microalgae and affects biomass and lipid yields of microalgae. In this study, the effect of nitrate on cell growth and lipid production and composition in Monoraphidium contortum, Tetraselmis suecica, and Chlorella minutissima was investigated. Nitrate affected the production of biomass and the production and composition of lipids of the three microalgae tested. Increasing the nitrate concentration in the culture medium resulted in increased biomass production and higher biomass productivity. Furthermore, increasing the nitrate concentration resulted in a reduction in lipid content and productivity in M. contortum; however, the opposite effect was observed in T. suecica andC. minutissima cultures. C. minutissima and M. contortum lipids contain high levels of oleic acid, with values ranging from 26 to 45.7% and 36.4 to 40.1%, respectively. The data suggest that because of its high lipid productivity (13.79 mg L-1 d-1) and high oleic acid productivity (3.78 mg L-1 d-1), Chlorella minutissima is a potential candidate for the production of high quality biodiesel.

Keywords: Chlorella minutissima • Monoraphidium contortum • Tetraselmis suecica • Biodiesel • Lipid production • Fatty acid composition © Versita Sp. z o.o.

1. Introduction

The growing global demand for energy, the increasing price of oil and its derivatives, the depletion of fossil fuels, the growing problem of air, soil, and water pollution caused by the extensive use of fossil fuels, and the problems pollution causes for human health and public security have prompted the search for energy sources that are renewable, sustainable, and environmentally

friendly. Biodiesel is an attractive source of renewable energy and is expected to play a major role in the global energy market in the future [1].

Biodiesel is a mixture of mono-alkyl esters of long chain fatty acids derived from vegetable oils or animal fats (htpp://www.astm.org). As a fuel, biodiesel is used in its pure state or is blended with petroleum-based diesel for conventional diesel engines. In addition, biodiesel can be used in the existing petroleum-based

E-mail: ecristianiu@yahoo.com.mx

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diesel infrastructure for storage and distribution. Biodiesel offers several advantages over petroleum-based diesel. For example, biodiesel is a renewable and biodegradable energy resource, it produces fewer toxic emissions (carbon monoxide, aromatic compounds, hydrocarbons, particulate matter, sulfur oxides, nitrogen oxides, and metals), it is less volatile and safer to transport, store and handle, and it increases efficiency, reduces wear, and extends engine life [2,3].

Currently, biodiesel is produced from vegetable oils (edible or inedible), waste oils, and animal fats. However, this practice has raised serious concerns in the international community regarding the production, price, and availability of food. Other concerns include the deforestation of large areas of land that could be used to grow oleaginous vegetables, the huge amounts of water needed for irrigation, and the inability of these biodiesel sources to meet current and future fuel demands [4,5]. A sustainable biodiesel industry needs alternative raw materials that can be obtained easily from alternative renewable and biodegradable sources, allowing continuous operation and avoiding the limitations described [6].

Currently, there is a consensus that microalgae are an alternative and sustainable source of lipids that can be used as a feedstock for biodiesel production. Microalgae are suitable for this purpose because they are photosynthetic microorganisms with a simple cellular structure and are easy to culture. Furthermore, microalgae grow in a variety of environments, their growth rate is 20 to 30 times faster than other sources of biofuels, they exhibit high photosynthetic efficiency, contribute greatly to the sequestration of atmospheric CO2, thereby mitigating climate change, can be harvested 365 days a year, can be grown in areas unsuitable for agriculture, livestock, industry, or tourism, require smaller volumes of water than oleaginous plants and can use water unsuitable for human consumption, their intracellular lipid content is high and the productivity of lipids per unit area is considerably higher than that of oleaginous plants [7-10].

Several studies have shown that a number of microalgae produce substantial amounts of lipids when grown under conditions of stress, such as the nutritional stress caused by nitrogen deficiency. Nitrogen is one of the most important nutrients for these organisms. Nitrate is the main nitrogen source for many microalgae and affects cell growth and lipid production [11,12].

Therefore, this study investigated the effect of nitrate on cell growth, nitrate consumption, lipid production, and the fatty acid composition of the lipids in the native microalgae Tetraselmis suecica, Monoraphidium contortum, and Chlorella minutissima.

2. Experimental Procedures

2.1 Microorganisms

The freshwater microalgae Chlorella minutissima and Monoraphidium contortum used in this study were isolated from Lake Xochimilco, Mexico, and were provided by the Department of Comparative Biology of the Science Faculty at the National Autonomous University of Mexico (UNAM, Universidad Nacional Autónoma de México), Mexico. The marine microalga Tetraselmis suecica TES1 was obtained from the Aquaculture Department at the Center for Scientific Research and Higher Education of Ensenada (CICESE, Centro de Investigación Científica y de Educación Superior de Ensenada), Baja California, Mexico.

2.2 Culture media

Bold's basal medium was used to cultivate the freshwater microalgae. The chemical composition of Bold's culture medium was as follows: NaNO3, 113 mg L-1; K2HPO4, 75 mg L-1; KH2PO4, 175 mg L-1; CaCl2 2H2O, 25 mg L-4; MgSO4 7H2O, 75 mg L-1; EDTA, 5 mg L-1; KOH, 3.1 mg L-1; FeSO47H2O, 0.498 mg L-1; concentrated H2SO4, 0.01 mL; H3BO3, 1.142 mg L-1; ZnSO47H2O, 0.882 mg L-1; MnCl24H2O, 0.144 mg L-1; MoO3, 0.071 mg L-1; CuSO45H2O, 0.157 mg L-1; and Co(NO3)26H2O, 0.049 mg L-1 [13].

For cultivating the marine microalga T. suecica, an artificial seawater medium with the following chemical composition was used: NaCl, 29.23 g L-1; KCl, 1.105 g L-1; MgSO47H2O, 11.09 g L-1;Tris base, 1.21 g L-1; CaCl22H2O, 1.83 g L-1; NaHCO3, 0.25 g L-1; NaNO3, 225 mg L-1; NaH2PO4 H2O, 15 mg L-1; Na2EDTA, 33.08 mg L-1; FeCl36H2O, 9.48 mg L-1; MnCl24H2O, 0.54 mg L-1; CoCl26H2O, 0.03 mg L-1; CuSO45H2O, 0.03 mg L-1; ZnSO47H2O, 0.069 mg L-1; Na2MoO4, 18 mg L-1; vitamin B1, 0.3 mg L-1; vitamin B12, 1.5 mg L-1; and biotin, 1.5 mg L-1 [14].

The microalgae were maintained at 25°C in vials and Petri dishes containing agar culture medium (Bold's basal liquid medium or artificial seawater medium containing 1.5% w/v bacteriological agar).

The inocula were prepared by transferring single colonies to 500-mL Erlenmeyer flasks containing 330 mL of culture media. The flasks were maintained at 25±1°C under continuous aeration with a sterile air flow of 1 vvm and constant white cool fluorescent lamp illumination (24 h) for 2 weeks. The light intensity was approximately 80 |jE m-2 s-1 and measured with a sensitive Hansatech Quantitherm light meter thermometer (Hansatech Instruments Ltd.). The cells were harvested by centrifugation at 3000*g for 15 minutes. The cell pellet was washed twice using sterile distilled water and was

resuspended in a small volume of culture medium. A sample of this cell suspension was used as the inoculum for the experiments performed in this study.

2.3 Culture conditions

The experiments to determine the influence of nitrate on cell growth, nitrate consumption, lipid production, and fatty acid composition of the microalgae tested in this study were performed in 500-mL Erlenmeyer flasks containing 330 mL of culture media with varying concentrations of nitrate. Three different initial concentrations of sodium nitrate were tested: 57, 113 and 225 mg L-1 (0.67, 1.33 and 2.65 mM NO3). Each flask was inoculated with a small volume of concentrated cell suspension to generate an initial biomass concentration of approximately 0.02 g L-1. The microalgal cultures were maintained under 1 vvm sterile continuous air flow at 25±1°C with a photoperiod of 16 h light and 8 h dark and a light intensity of approximately 80 pE m-2 s-1 (provided by a neon cool white fluorescent lamp).

The samples were collected at different incubation times, and the biomass and residual nitrate concentrations were determined. The lipid content and fatty acid composition of the microalgae were evaluated at the early stationary growth phase, when cells growth became plateau.

The maximum specific growth rate (jmax, d-1), duplication time (td, d) and biomass productivity (PX, g L-1 d-1) of every microalga was estimated as follows:

where X1 and X2 are biomass concentrations at time 1 (t1) and time 2 (t2) in the exponential growth phase, respectively; XO is the initial biomass concentration at time to=0 d, Xf is the biomass concentration at time t, and tf is the cultivation time at the beginning of the stationary growth phase.

Nitrate consumption performance of each microalga was evaluated according to two criteria: removal efficiency [E, %] and volumetric rate [RV, mg L-1 d-1] of nitrate removal, which were calculated as follows:

where (NaNO3)/ is initial sodium nitrate concentration at time t=0 d, (NaNO3)t is residual sodium nitrate concentration at time t=tt, and tt is the cultivation time at which nitrate was completely consumed or the total incubation time for those experiments in which nitrate was not completely consumed.

Lipid productivity (PL, g lipid L-1 d-1) was expressed as follows:

where Y (g lipid g-1 biomass) is the lipid content of microalgal cells at the beginning of the stationary phase of growth.

All experiments and analysis in this study were carried out in triplicate and average values are reported herein.

2.4 Analytical methods

2.4.1 Biomass concentration

Biomass concentration was determined by measuring dry cell weight. Culture samples were filtered through pre-weighed 1.6 jm filters (Whatman GF/A), which were washed twice with sterile distilled water and subsequently dried at 90°C until constant weight was attained. The obtained filtrates were used to determine residual nitrate concentration and pH.

2.4.2 Nitrate concentration

Nitrate concentration was determined using the cadmium reduction method (NitraVer®5, Hach) in accordance with the procedure described in the Hach Water Analysis Handbook [15]. The absorbance was read at 500 nm in a Genesys™ 10 UV/Visible spectrophotometer (Thermo Electron).

2.4.3 Lipid extraction

The early stationary growth phase is the critical stage for evaluating the accumulation of lipids in microalgae [16]. Therefore, in this study, the determination of lipid content of the 3 microalgal strains was performed using cells in the early stationary phase of growth.

The microalgal cells were separated from culture medium by centrifugation (3000*g for 15 min). The cell pellets were washed twice using deionized water and then dried by lyophilization (Heto Drywinner, Denmark). A sample (30 mg) of the dry biomass was mixed with 15 mL of a methanol:chloroform solution (2:1, v/v) and was sonicated for 1 h. Next, 5 mL of chloroform and 5 mL of a 1% NaCl (w/v) solution were added to the sonicated suspension. The resulting mixture was

centrifuged at 3000xg for 10 min, and the chloroform phase containing the total lipids was recovered. Finally, the chloroform was evaporated using a rotary evaporator (Buchi, Switzerland) and the oil content was measured gravimetrically and expressed as a dry weight percentage [17].

2.4.4 Fatty acid analysis

Esterification of fatty acids into methyl esters was carried out following the method described by O'Fallon et al. [18] with slight modification. Samples of the extracted oil (10 mg) were placed into 16x125 mm screw-cap Pyrex tubes to which 0.5 mL of BF3 in methanol (12%, w/v) and 100 |jL of heptadecanoic fatty acid (0.1 mg) as an internal standard were added. The tubes were incubated in a water bath at 90°C for 20 min with shaking. After cooling, 0.5 mL of hexane and 1.0 mL of distilled water were added, and the tubes were vortex-mixed. After centrifugation, the hexane layer containing the fatty acid methyl esters (FAMEs) was placed into gas chromatography vials.

The separation and quantification of FAMEs was carried out using a mass spectrophotometer (Agilent Serie 5975C) coupled to a gas chromatograph (Agilent 6850 Series II Network System). Gas chromatograph is equipped with a flame ionization detector and a capillary column (Agilent 19091s-433E; 30 m * 250 |jm x 0.25 jm). The FAME samples were injected into the column via an automated split injector. The split ratio was 55.7:1. The initial oven temperature was 150°C, held for 2 min, subsequently increased to 200°C at a rate of 5°C/min, and then moved to 260°C at a rate of 3°C/min. The temperature for injector and detector were set at 220 and 290°C, respectively. Helium was used as the carrier gas, with a constant flow of 1.0 mL min-1.

3. Results and Discussion

3.1 Effect of nitrate on cell growth

Figure 1 shows the growth curves of C. minutissima, T. suecica, and M. contortum at the 3 initial nitrate concentrations tested. The time required for the microalgae to reach the stationary growth phase increased with the increase in the initial nitrate concentration of the culture medium. In addition, the biomass concentration increased in all 3 microalgal cultures when the initial nitrate concentration was increased. These findings are in agreement with similar reports using Dunaliella tertiolecta [1], Scenedesmus sp. [19], Chlorella vulgaris UMT-M1, and Chlorella sorokiniana KS-MB2 [16].

It is apparent from Figure 1 that the maximum biomass concentrations of C. minutissima were 0.34, 0.63 and 0.94 g L-1, and those of M. contortum were 0.26, 0.44 and 0.80 g L-1, at sodium nitrate concentrations of 57, 113 and 225 mg L-1, respectively. Similarly, an increase in the maximum biomass concentration of the marine microalga T. suecica was observed when the initial nitrate concentration was increased, reaching values of 0.48, 0.68, and 0.90 g of biomass L-1 at the sodium nitrate concentrations of 57, 113, and 225 mg L-1, respectively.

Figure 1. Changes in biomass concentration during batch cultures of M. contortum (A), T. suecica (B), and C. minutissima (C) at the initial NaNO3 concentrations of 57 (▲), 113 (♦) and 225 (■) mg L-1.

Nitrate is an essential macronutrient required for the synthesis of amino acids, proteins, nucleic acids, and other nitrogen-containing compounds that form part of the biomass of microalgae, which may explain the increase in biomass concentration that is observed following the increase in the initial nitrate concentration. Consequently, the increased nitrate availability in the culture medium produced an increase in the cell mass.

Table 1 shows the values for the maximum specific growth rate (Jmax) and the duplication time (td) during the exponential growth phase of C. minutissima, M. contortum, and T. suecica. Notably, for the 3 microalgae tested, the maximum specific growth rate increased and the duplication time decreased with increased initial concentrations of sodium nitrate. However, the statistical analysis revealed that differences in the specific growth rates and duplication times in each strain were not significant (P>0.05) at the initial concentrations of nitrate tested; however, the specific growth rate and duplication times were statistically different (P<0.05) between the different strains.

At the 3 initial nitrate concentrations tested, T. suecica exhibited the highest maximum specific growth rate (1.17-1.20 d-1), followed by C. minutissima (0.54-0.60 d-1) and M. contortum (0.23-0.32 d-1). Therefore, the smallest duplication time corresponded to T. suecica, followed by C. minutissima and M. contortum. The maximum specific growth rates of T. suecica obtained in this study are consistent with that reported by Carballo-Cardenas et al. [20] (|Jmax = 0.05 h-1 = 1.2 d-1).

Table 1 shows the overall biomass productivity results for the 3 microalgal strains at the 3 different nitrate concentrations tested. Clearly, the microalgal cell productivity increased as the initial concentration of nitrate increased. The highest biomass productivity (0.04 g L-1 d-1) was obtained with T. suecica and C. minutissima at 225 mg L-1 sodium nitrate concentration.

The data suggest that although the maximum specific growth rate (Mmax) and the duplication time of the microalgae during the exponential growth phase were not affected by the initial nitrate concentration, the maximum biomass concentration and overall biomass productivity were strongly dependent on the concentration of this macronutrient.

3.2 Effect of initial nitrate concentration on its consumption

As discussed previously, nitrates are the main source of nitrogen for many microalgae, and the consumption of nitrate is closely related to cell growth.

Moreover, microalgae have attracted attention because of their ability to remove nitrate from water and their potential as biodiesel producers [19]. Therefore, from a sustainable development perspective, it is important that the microalgae used for biodiesel production can consume all or most of the nitrate in the growth medium.

Figure 2 displays the nitrate consumption profiles of C. minutissima, M. contortum, and T. suecica for the 3 different initial sodium nitrate concentrations tested. As the initial nitrate concentration in the culture medium increased, the microalgae required more time to consume the nitrate completely or almost completely. A similar pattern was observed in Scenedesmus sp. LX1 cultured in modified BG11 medium at different initial nitrate concentrations [19]. In contrast, as expected, the overall nitrate consumption rates increased as the initial concentration of nitrate in the culture medium increased. The highest nitrate consumption rates were observed in T. suecica and C. minutissima (Table 2), which also produced the highest cell densities.

At the 3 nitrate concentrations, the overall nitrate consumption efficiencies exhibited by T. suecica and C. minutissima were 100%, whereas the consumption

M. contortum

T suecica

C. minutissima

Initial NaNO3 concentration [mg L-1]

Maximum specific growth rate [d-1]

Duplication time [d]

Biomass productivity

[g L-1 d-1]

113 225

0.23 ± 0.01 0.26 ± 0.01 0.32 ± 0.01 1.17 ± 0.03 1.19 ± 0.02 1.20 ± 0.02 0.54 ± 0.04 0.57 ± 0.02 0.60 ± 0.04

3.00 ± 0.09 2.65 ± 0.11 2.14 ± 0.14 0.59 ± 0.03 0.58 ± 0.01 0.57 ± 0.01 1.29 ± 0.01 1.22 ± 0.04 1.16 ± 0.10

0.01 ± 0.001 0.02 ± 0.001 0.03 ±0.001 0.02 ± 0.001 0.03 ± 0.001 0.04 ± 0.001 0.01 ±0.005 0.03 ± 0.005 0.04 ± 0.002

Table 1. Maximum specific growth rate, duplication time and microalgal biomass productivity at different initial sodium nitrate concentrations.

efficiencies of M. contortum were 100%, 100%, and 96.53% at the initial sodium nitrate concentrations of 57, 113, and 225 mg L-1, respectively (Table 2). The data clearly demonstrate that the 3 microalgal strains used in this study efficiently remove nitrate from aqueous solutions.

3.3 Effect of nitrate on lipid production by microalgae

Evidently the occurrence and extent to which microalgal lipids are produced is species- and even strain-specific, and ultimately controlled by the genetic make-up of individual organisms [21]. Depending on the species and the physicochemical cultivation conditions, the microalgal lipid content varies from approximately 1 to 85% of the dry cell weight [7,22,23]. The accumulation of reserve materials (carbohydrates and lipids) in microalgae usually occurs during periods of environmental stress. Nitrogen is the single most critical nutrient affecting lipid metabolism in algae [21] and the nutritional stress caused by nitrogen deficiency is an efficient strategy for increasing the lipid content of microalgae. However, because the performance of microalgae facing nitrogen deficiency is highly variable, it is impossible to establish a general trend among microalgal species [23]. Thus experiments are required for individual microalgal strains, in order to determine the influence of nitrogen limitation on lipid accumulation. In this study, we determined that the lipid content of M. contortum increased significantly (P<0.05) from 9.6 to 32.6% when the sodium nitrate content decreased from 225 to 57 mg L-1 (Table 3). This trend for lipids to accumulate in response to nitrogen deficiency has been observed among numerous species or strains of microalgae, including Chlamydomonas reinhardtii, Scenedesmus subspicatus [24], Nannochloropsis oculata [12], Nannochloropsis sp. [23,25], Tetraselmis suecica [23], Chlorella vulgaris [2,12,26,27], Chlorella sp. [28], Chlorella minutissima [29], Phaeodactylum tricornutum [30], Dunaliella tertiolecta [1 ], and Neochloris oleabundans [25].

Figure 2. Variation in residual sodium nitrate concentration during batch cultures of M. contortum (A), T. suecica (B), and C. minutissima (C) at the initial NaNO3 concentrations of 57 (▲), 113 (♦) and 225 (■) mg L-1.

M. contortum T. suecica C. minutissima

Initial NaNO3 concentration [mg L-1] 57 113 225 57 113 225 57 113 225

Nitrate consumption efficiency [%] 100 100 96.53 ± 1.3 100 100 100 100 100 100

Nitrate consumption rate [mg L-1 d-1] 2.38 ± 0.11 5.23 ± 0.16 6.4 ± 0.27 7.0 ± 0.14 11.3 ± 0.34 16.07 ± 0.64 8.0 ± 0.45 11.3 ± 0.28 12.14 ± 0.48

Table 2. Overall efficiency and rate of nitrate consumption at different initial sodium nitrate concentrations.

M. contortum T. suecica C. minutissima

Initial NaNO3

concentration 57 113 225 57 113 225 57 113 225

[mg L-1]

Lipid content [%, w/w] 32.6 ± 1.11 28.2 ± 0.96 9.6 ± 1.21 8.0 ± 0.23 6.38 ± 0.15 17.15 ± 0.49 22.7 ± 0.92 36.6 ± 0.66 36 ± 0.88

Lipid productivity [mg L-1 d-1] 4.89 ± 0.16 4.79 ± 0.10 2.78 ± 0.35 1.70 ± 0.04 1.92 ± 0.04 7.04 ± 0.20 2.96 ± 0.12 9.35 ± 0.16 13.79 ± 0.34

Table 3. Lipid content and productivity at different initial nitrate concentrations.

The increase in microalgal lipid content, corresponding to a decrease in nitrate in the culture medium has been attributed to the fact that when nitrogen is limited, photosynthetically derived energy (normally channeled into producing more cells and therefore more proteins) is in part diverted into making storage products such as carbohydrates and lipids, most of which lack nitrogen [31]. This implies that when nitrogen is limited, algal growth slows down and thus there is no need for the synthesis of new membrane compounds, proteins, nucleic acids, and other nitrogen-containing compounds that form part of the microalgal biomass; instead the cells divert and accumulate lipids. Likewise, nitrogen limitation tends to bring on the following three changes: a decrease in the glycolipid and phospholipid content of thylakoid membrane [19,32,33], activation of acyl hydrolase and stimulation of phospholipid hydrolysis. These changes may increase the intracellular content of fatty acid acyl-CoA. Meanwhile, nitrogen limitation may activate diacylglycerol acyltransferase, which converts acyl-CoA to triglyceride (TAG). Thus, nitrogen limitation may increase both lipid and TAG content in microalgal cells [19]. Lipids produced under these circumstances function as a carbon and energy reserve and protect the organism against photo-oxidative stress [34].

In contrast, the low initial concentration of sodium nitrate (57 mg L-1) did not produce a favorable effect on lipid accumulation in T. suecica, producing approximately 8% lipids under these culture conditions. The lipid accumulation in T. suecica increased significantly (P<0.05) when an initial sodium nitrate concentration of 225 mg L-1 was used, and the lipid content reached approximately 17.15% (Table 3). Similarly, the lipid content in C. minutissima increased significantly (P<0.05) from 22.7 to 36.6% when the initial concentration of sodium nitrate increased from 57 to 113 mg L-1, respectively. However, a further increase in the sodium nitrate concentration to 225 mg L-1 did not affect (P>0.05) the lipid accumulation in this species (Table 3).

The increase in lipid content which takes place with the increase in nitrate availability has been attributed

to an increase in the activity of acetyl-CoA carboxylase (ACCase), causing an increase in lipid production [35,36]. As with other algae, including Isochrysis zhangjiangensis, Cyclotella cryptica [35], and Isochrysis galbana [36], the activity of acetyl-CoA carboxylase or other key enzymes of T. suecica and C. minutissima may be enhanced under non-limiting nitrogen conditions, resulting in an increase in lipid production.

An increase in lipid content, corresponding to the increase in nitrogen concentration has also been observed in the case of Ellipsoidion sp. [37], Isochrysis zhangjiangensis [35], Tetraselmis suecica [31], Chlorella sorokiniana and Chlorella vulgaris [16], among others.

The data demonstrate that C. minutissima and M. contortum accumulate large amounts of lipids, approximately 36% and 32.6%, when the initial nitrate concentration are 113-225 and 57 mg L-1, respectively. These lipid levels are higher than those reported for other oleaginous microalgae, such as Botryococcus sudeticus [38], Scenedesmus obliquus, Scenedesmus dimorphus, Dunaliella bioculata, Dunaliella salina [25], Chlorella protothecoides, Chlorella sorokiniana [39], Chlorella sp., Crypthecodinium cohnii, Dunaliella primolecta, and Isochrysis sp. [22], among others.

Moreover, the volumetric productivity of lipids (the mass of lipid produced per unit volume of the microalgal broth per day) depends on the algal specific growth rate, the amount of algal biomass produced, and the lipid content of the biomass. The economics of the biodiesel production process depends on lipid productivity. Therefore, microalgae that exhibit high lipid productivity are desirable. Despite the importance of this feature, few studies have reported lipid productivity data for microalgae.

Table 3 shows the lipid productivity of T. suecica, M. contortum, and C. minutissima. In T. suecica and C. minutissima the lipid productivity increased with the increase in the initial nitrate concentration. This increase in lipid productivity is attributed to the fact that increasing nitrate concentration in the culture medium produced an increase in biomass productivity and lipid content (Tables 1 and 3).

In contrast, in M. contortum, lower concentrations of nitrate in the culture medium increased lipid accumulation (32.6%) and lipid productivity (4.89 mg L-1 d-1). As lipid productivity depends directly on biomass productivity and lipid content (equation 6), lipid productivity of M. contortum decreased significantly when initial nitrate concentration increased. This is because lipid content was found to decrease as the nitrate concentration was increased. Therefore, the nitrate level in the culture medium produces the opposite effects on the content and the productivity of lipids in M. contortum in comparison with T. suecica and C. minutissima.

At the higher sodium nitrate concentration tested (225 mg L-1), the lipid productivity of C. minutissima (13.79 mg L-1 d-1) was substantially higher than that of the other microalgal strains. The high lipid content and lipid productivity in C. minutissima suggest that this microalga is potentially useful for biodiesel production.

3.4 Effect of nitrate on fatty acid composition

The important properties of biodiesel, such as density, kinematic viscosity, cetane number, oxidation stability, cold flow properties, and heat of combustion, among others, are highly dependent on the fatty acid composition of the microalgal lipids. In addition, the fatty acid composition depends on several factors, including the strain of microalgae used and the physicochemical conditions for cultivation. This study evaluated the effect of nitrate on the fatty acid composition of the 3 microalgal strains at the early stationary growth phase, when the cell growth reached a plateau.

The fatty acid composition of the microalgal lipids was analyzed following the extraction and transesterification of the lipids and the analysis of the FAMEs using MS-GC. Importantly, biodiesel is a mixture of FAMEs derived from lipids through a transesterification process. However, not all lipids can be converted to FAMEs, which are the chemical ingredients of biodiesel. Therefore, the measurement of FAMEs in microalgal biomass is a direct indication of the amount of lipids suitable for the production of biodiesel [4].

Moreover, it is known that oleic acid is a major component of biodiesel and an important indicator of biodiesel quality [40]. It has been reported that lipids rich in oleic acid exhibit properties that make them suitable for fuel, including their ignition quality, appropriate heat of combustion, cold filter plugging (CFPC), oxidative stability, viscosity, and lubricity [41]. Therefore, microalgae that exhibit high oleic acid productivity are desirable.

Tables 4 and 5 show the fatty acid content and the fatty acid productivity of the 3 microalgal strains at the three sodium nitrate concentrations tested, respectively.

At initial nitrate concentrations of 57 and 113 mg L-1, the main fatty acids found in the M. contortum oils consisted of oleic (C18:1) and palmitic (C16:0) acids, with values of 36.4 and 40.1% and 27.3 and 24%, respectively. The volumetric productivities of oleic acid and palmitic acid were also the highest at these nitrate concentrations, with values for oleic acid of 1.78 and 1.92 mg L-1 d-1, and for palmitic acid of 1.33 and 1.15 mg L-1 d-1. Contrastingly, linolenic fatty acid (C18:3) predominated when the microalga was cultured at an initial concentration of 225 mg L-1 sodium nitrate, with a content of 52.6% and a productivity of 1.46 mg L-1 d-1.

In the case of T. suecica, palmitic acid (C16:0) was the main fatty acid found at the three concentrations of nitrate tested, with values for content and productivity ranging from 20 to 28.3%, and 0.48 to 1.41 mg L-1 d-1, respectively. The second most abundant fatty acid was apparently directly related to the initial concentration of sodium nitrate in the medium, consisting of linoleic acid (C18:2) at 57 and 113 mg L-1 sodium nitrate and oleic acid (C18:1) at 225 mg L-1 sodium nitrate. The linoleic acid content and productivity at 57 and 113 mg L-1 sodium nitrate was 24.3 and 22.4% and 0.41 and 0.43 mg L-1 d-1 respectively, and those for oleic acid at 225 mg L-1 were 14.3% and 1.0 mg L-1 d-1.

When C. minutissima was grown in culture medium at an initial concentration of 57 mg L-1 sodium nitrate, the primary fatty acids in the oils were oleic acid (C18:1) and palmitic (C16:0) acid at 45.7 and 41.6%, respectively. At higher concentrations of sodium nitrate (113 and 225 mg L-1), the predominant fatty acids were isopalmitic acid (C16:0, an isomer of palmitic acid) and oleic acid (C18:1). The oleic acid and the 9,15-octadecanoic acid were the only fatty acids detected at the three initial nitrate concentrations tested, and their productivity increased as the sodium nitrate concentration increased. The highest oleic acid productivity (3.78 mg L-1 d-1) was obtained with C. minutissima at an initial nitrate concentration of 225 mg L-1 (Table 5).

The data clearly demonstrate that the fatty acid composition of the lipids in M. contortum, T. suecica, and C. minutissima is dependent on the nitrate concentration in the culture medium. In addition, under conditions of nitrate limitation in the medium (57 mg NaNO3 L-1), the microalgae tested in this study accumulated larger amounts of saturated fatty acids with 16 carbons and unsaturated fatty acids with 18 carbons, concurring with reports for other microalgae [8,23,42].

In this study, the highest amounts of oleic acid were detected in M. contortum and C. minutissima. The oleic acid content of the M. contortum and C. minutissima strains used in this study is higher than reported for Ourococcus multisporus YSW08, Scenedesmus

CJI CO

M. contortum I suecica C. minutissima

Initial NaN03 concentration [mg L1] 57 113 225 57 113 225 57 113 225

Laurie acid C12:0 nd nd 19.3 ± 1.5 nd nd nd nd nd nd

9-Laurolelc acid C12:1 nd nd 1.3 ± 0.1 nd nd nd nd nd nd

Myrlstolelc acid C14:1 nd nd nd 6.1 ±0.2 6.3 ± 0.2 10 ± 0.4 nd nd nd

Palmitic acid C16:0 27.3 ± 2.1 24.0 ± 1.8 nd 28.3 ± 1.1 28.0 ± 1.1 20 ± 0.8 41.6 ± 1.7 nd nd

Isopalmltlc acid C16:0 nd nd nd nd nd nd nd 29.2 ± 1.2 28.6 ± 1.2

7-Palmltolelc acid C16:1 9.1 ± 0.7 1.8 ± 0.1 nd nd nd nd nd nd nd

Palmltolelc acid C16:1 nd nd 1.96 ± 0.2 nd nd nd nd nd nd

7,10-Hexadecadlenolc acid C16:2 nd nd nd nd nd nd nd nd 2.1 ± 0.1

Stearic acid C18:0 nd nd nd nd nd 5.7 ± 0.2 nd nd nd

Isostearlc acid C18:0 9.1 ± 0.7 1.3 ± 0.1 nd nd nd nd nd nd nd

Oleic acid C18:1 36.4 ± 2.8 40.1 ± 3.1 nd 8.7 ± 0.3 9.5 ± 0.4 14.3 ± 0.6 45.7 ± 1.9 26 ± 1.1 27.4 ± 1.1

Llnolelc acid C18:2 9.1 ± 0.7 5.2 ± 0.4 6.0 ± 0.5 24.3 ± 1.0 22.4 ± 0.9 8.6 ± 0.3 nd nd nd

9,15-octadecanolc acid C18:2 nd nd nd nd nd nd 12.7 ± 0.5 17 ± 0.7 16.4 ± 0.7

Llnolenlc acid C18:3 nd nd 52.6 ± 0.4 6.5 ± 0.3 6.9 ± 0.3 10 ± 0.4 nd 12.1 ± 0.5 11.0 ± 0.5

Gondolc acid C20:1 nd 2.7 ± 0.2 nd nd nd nd nd nd nd

Araquldonlc acid C20:4 nd nd nd 3.9 ± 0.2 2.3 ± 0.1 2.9 ± 0.1 nd nd nd

Elcosapentaenolc acid C20:5 nd nd nd 3.0 ± 0.1 2.3 ± 0.1 4.3 ± 0.2 nd nd nd

Docosahexaenolc acid C22:5 nd 5.9 ± 0.4 6.7 ± 0.5 nd nd nd nd 5.9 ± 0.2 6.7 ± 0.3

Saturated fatty acids 36.4 25.3 19.3 28.3 28.0 25.7 41.6 29.2 28.6

Monounsaturated fatty acids 45.5 44.6 3.26 14.8 15.8 24.3 45.7 26.0 27.4

Polyunsaturated fatty acids 9.1 11.1 65.3 37.7 33.9 25.8 12.7 35.0 36.2

Table 4. Fatty acid compositions (wt.p %) of M. contortum, C. minutissima and I suecica oils. nd = not detected

M. contortum I suecica C. minutissima

Initial NaN03 concentration [mg L1] 57 113 225 57 113 225 57 113 225

Laurie acid C12:0 nd nd 0.54 ± 0.04 nd nd nd nd nd nd

9-Lauroleic acid C12:1 nd nd 0.04 ± 0.002 nd nd nd nd nd nd

Myrlstolelc acid C14:1 nd nd nd 0.10 ± 0.001 0.12 ± 0.02 0.70 ± 0.03 nd nd nd

Palmitic acid C16:0 1.33 ± 0.09 1.15 ± 0.08 nd 0.48 ± 0.15 0.54 ± 0.03 1.41 ± 0.05 1.17 ± 0.04 nd nd

Isopalmitlc acid C16:0 nd nd nd nd nd nd nd 2.73 ± 0.11 3.98 ± 0.16

7-Palmitoleic acid C16:1 0.44 ± 0.03 0.09 ± 0.07 nd nd nd nd nd nd nd

Palmltolelc acid C16:1 nd nd 0.05 ± 0.004 nd nd nd nd nd nd

7,10-Hexadecadlenoic acid C16:2 nd nd nd nd nd nd nd nd 0.29 ± 0.01

Stearic acid C18:0 nd nd nd nd nd 0.40 ± 0.03 nd nd nd

Isostearlc acid C18:0 0.44 ± 0.03 0.06 ± 0.005 nd nd nd nd nd nd nd

Oleic acid C18:1 1.78 ± 0.05 1.92 ± 0.06 nd 0.15 ± 0.005 0.18 ± 0.08 1.01 ± 0.03 1.28 ± 0.04 2.43 ± 0.09 3.78 ± 0.09

Linoleic acid C18:2 0.44 ± 0.03 0.25 ± 0.02 0.17 ± 0.01 0.41 ± 0.007 0.43 ± 0.01 0.60 ± 0.02 nd nd nd

9,15-octadecanoic acid C18:2 nd nd nd nd nd nd 0.35 ± 0.01 1.59 ± 0.06 2.28 ± 0.08

Linolenic acid C18:3 nd nd 1.46 ± 0.1 0.11 ± 0.003 0.13 ± 0.001 0.70 ± 0.03 nd 1.13 ± 0.04 1.53 ± 0.06

Gondoic acid C20:1 nd 0.13 ± 0.01 nd nd nd nd nd nd nd

Araquidonlc acid C20:4 nd nd nd 0.07 ± 0.002 0.04 ± 0 002 0.20 ± 0.01 nd nd nd

Elcosapentaenoic acid C20:5 nd nd nd 0.05 ± 0.002 0.04 ± 0.002 0.30 ± 0.01 nd nd nd

Docosahexaenolc acid C22:5 nd 0.28 ± 0.02 0.19 ± 0.01 nd nd nd nd 0.55 ± 0.02 0.93 ± 0.04

Table 5. Fatty acid productivity (mg L1 d"1) of M. contortum, C. minutissima and I suecica. nd = not detected

obliquus YSR05, S. obliquus YSR01 [41], Botryococcus braunii [43], Chlorella humicola [8], and Chlorella minutissima UTEX 2219 [29], among other microalgae.

Results show that Chlorella minutissima exhibited the highest lipid content (36%), lipid productivity (13.79 mg L-1 d-1) and oleic acid productivity (3.78 mg L-1 d-1) (Tables 4 and 5). These results indicate that C. minutissima is a high quality feedstock for biodiesel production.

4. Conclusions

The present study clearly indicates that nitrate level in the culture medium affects biomass and lipid productivities of the oleaginous microalgae Chlorella minutissima, Tetraselmis suecica and Monoraphidium contortum. Fatty acid composition of microalgal oil is also affected by nitrate level. The C. minutissima strain

used in this work presents high lipid content, lipid productivity, and oleic acid productivity, making this microalga a potential candidate for the production of high quality biodiesel.

Acknowledgements

The authors gratefully acknowledge the support provided by the scientific team of the Central Laboratories of Spectroscopy, and Biotechnology and Molecular Biology at National School of Biological Sciences, National Polytechnic Institute, as well as the financial support provided by the Secretariat of Posgraduate Studies and Research, National Polytechnic Institute. T.L. Villegas-Garrido and E. Cristiani-Urbina are fellow holders of a grant from the Commission for Support and Operation of Academic Activities, National Polytechnic Institute, Mexico City, Mexico.

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