Effects of Irradiance and Different Nitrogen and Carbon Concentrations on the Minerals Accumulation in Scenedesmus obliquus Biomass

Microalgae are capable of absorbing and concentrating constituent elements that have a wide variety of applications in agriculture, food industry, and medicine. Microalgae chemical composition change according to internal and external factors. In this study, the effect of irradiance, sodium nitrate and sodium acetate concentration on the accumulation of essential minerals in Scenedesmus obliquus biomass were evaluated using 23 factorial screening designs. The simultaneous effect of the three experimental factors was studied using three levels for each parameter (irradiance: 36.71, 69.50, 102.30 μE m-2 s-1, sodium nitrate: 0.27, 44.00, 87.73 g L-1 and sodium acetate: 0.00, 2.50, 5.02 g L-1). The response variables were the minerals concentration of Na, K, Ca, Mg, Fe, Zn and Mn. Results show that each mineral has an optimal operation condition in order to improve its concentration in the microalgae biomass. A signifi cant interaction between the variables was observed, which has direct effects on the minerals accumulation in the microalgae biomass. Under these conditions, the maximum concentration of K (1515.77 [mg (100gdw) -1]), Ca (2744.24 [mg (100gdw) -1]), Mg (9697.65 [mg (100gdw) -1]), Fe (2932.42 [mg (100gdw) -1]), Mn (38.48 [mg (100gdw) -1]), Zn (324.00 [mg (100gdw) -1]) and the minimum concentration of Na (5607.20 [mg (100gdw) 1]) were obtained from the microalga biomass. Thus, Scenedesmus obliquus biomass was characterized as good essential mineral source and confi rmed to be potentially valuable ingredient for utilization in the food industry. ABSTRACT

Minerals have diff erent functions in the human body, such as muscle function, nerve stimulation, enzymatic and hormonal activities, and oxygen transport. For example, Sodium (Na) and Potassium (K) both are regulating osmotic pressure. Calcium (Ca) and Magnesium (Mg) are key functional components of the tissue and skeleton [3]. Iron (Fe), is an essential mineral that plays an important role in many metabolic processes such as synthesis of haemoglobin and myoglobin (oxygen transport proteins); oxidationreduction processes [4]. Zinc (Zn) mainly participates in regulation of growth and diff erentiation cellular and it is an important co-factor in many enzymes [5]. Manganese (Mn) is a trace mineral of fundamental importance to human health, is an essential component of the antioxidant enzymes, involved in the formation of connective tissue, bones and nerve function [6]. Among mineral insuffi ciencies, defi ciencies in Fe and Zn are reported as highly prevalent nutritional problems around the world, aff ecting mainly developing country. Iron and zinc defi ciency may lead to slow cognitive and skeletal development and immunodefi ciency disorders [7]. Similarly, low calcium intake is associated with chronic diseases such as osteoporosis, colon cancer, hypertension and obesity [8]. However, an excess in the intake of other minerals, such as sodium, may trigger various body disorders, mainly cardiovascular disease and stroke. So, a balanced consumption of minerals is important for the proper functioning of the body. In this sense, dietary variation and/or supplementation are essential. The enrichment of food with natural source with high mineral content, such as microalgae, can be useful in avoiding the intake of supplementation or pharmaceutical products.
Nowadays, microalgae have numerous commercial applications in food and animal feed. For example, in food, microalgae are incorporated in pasta, sweet snack bars and drinks, as a natural nutritional supplement, enhancing its nutritional value [9].
Microalgae production is highly sustainable and has certain advantages over the use of other microorganisms due to its high productivity, fast growth, CO 2 absorption from the atmosphere, growth in extreme environmental conditions, and do not need agricultural land [10,11].
Microalgae biotechnology allows controlling cultivation conditions in order to production of many compounds of biological interest. There are several parameters that aff ect the microalgae growth and therefore its nutritional value: distribution and intensity of light within the bioreactor, temperature, nutritional composition of the culture media, among others [2,12]. According to the composition of the culture media, there are studies that refer the eff ect of sodium acetate, as a source of carbon, on the production microalgae biomass. Cordero, et al. [13], studied the eff ect of sodium acetate concentration on Chlorella sorokiniana biomass production. They observed a 20% increase in biomass when the sodium acetate concentration varied between 0 to 40 mM, while at higher levels of sodium acetate (50 and 60 mM), not observed changes in the biomass production.
Nitrogen is another nutrient that aff ects the growth and composition of microalgae. Microalgae species diff er in its nitrogen requirements and their assimilation kinetics [14].
Light is another factor that aff ects the microalgae production. Nutrient assimilation responds to the free energy gradient between inside and outside of cell. Thus, limitations in cellular energy can limit the assimilation of nutrients and consequently cell growth [14]. Changes in the nutritional composition of the culture medium as well as culture conditions induce stress in microalgae cultures, causing modifi cations in cell physiology and biochemical composition [15]. Therefore, it is important to determine both, nutritional composition optimum and culture conditions optimum for the achievement of high yields of microalgae with valuable metabolites.
This study was designed to determine the best combination of the variables sodium nitrate, sodium acetate and irradiance on the Scenedesmus obliquus culture in order to maximize and minimize the accumulation of essential minerals, and thus contribute to its possible application in formulated food in order to reduce the essential minerals defi ciency in a natural form.

Microalga inoculum
The Scenedesmus obliquus microalga (IOAC081F) was isolated from Embalse Salto Grande, a region of North-East Argentina [16]. The inoculum was cultured in batch mode, using Allen & Arnon culture medium enriched with 0.850 g L -1 of NaNO 3 . The culture was maintained in exponential growth phase at temperature of 25 ± 1°C, aerated at 0.20 v v -1 min -1 , a pH = 8.0 ± 0.1, a light intensity of 150 μE m -2 s -1 and a 12:12 h light/dark cycle.

Obtaining microalgal biomass: Culture media and operating conditions
The microalgal biomass obtaining was carrying out in two stages. Illumination was performed using a circadian cycle (12:12 light/dark). These operating conditions were maintained until reaching the stationary phase.

Experimental design
The central composite design was performed using Statgraphics Centurion XVI software. The three studied factors were irradiance (μE m -2 s -1 ), sodium nitrate concentration (g L -1 ) and sodium acetate concentration (g L -1 ) which were evaluated at three diff erent levels, using a

Sample preparation and determination of minerals
Lyophilized samples (~100 mg) were weighed in triplicate and digested using a Kjeldahl digester. For digestion, 5 mL of HNO 3 70% concentrated (Merck) was used, the temperature was maintained at 80 º C for 30 min.

Initial mineral concentration in the microalgae biomass
The initial mineral concentration in Scenedesmus obliquus cells is presented in

Effect of experimental factors on the Potassium (K) concentration
The  Administration [20], which could be achieved with a daily consumption of 132 g of Scenedesmus obliquus, produced under the indicated conditions. Otherwise, a daily intake of 510 g of Scenedesmus obliquus will be necessary. In other cases, a daily intake of approximately 150g of Spirulina platensis or 4000 g of Chlorella vulgaris will meet K requirements [18].

Effect of experimental factors on the Calcium (Ca) concentration
The  (Figure 4).    The recommended daily intake for Ca is 1000 mg per day for adults [20]. Therefore, a daily intake of approximately 36 g of Scenedesmus obliquus, produced under the indicated conditions, will meet Ca requirements for an adult.

Under these conditions the Ca concentration in
Otherwise, a daily intake of 37 g of Scenedesmus obliquus will be necessary.
In other cases, a daily intake of approximately 113 g of Spirulina platensis or 168 g of Chlorella vulgaris will meet Ca requirements [18]. For the Mg concentration, the eff ects of sodium nitrate concentration on its quadratic component (factor AA), sodium nitrate concentration-sodium acetate concentration interaction (Factor AB), sodium nitrate concentrationirradiance interaction (Factor AC) and sodium acetate concentration-irradiance interaction (Factor BC) were signifi cant. The eff ects of each factor and their interaction, as well as statistical signifi cance, are reported in fi gure 5.

Effect of experimental factors on the Magnesium (Mg) concentration
The signifi cance of sodium nitrate concentration (factor A) with the other factors (B and C) on Mg levels in the microalgae biomass shows that the A, B and C factors have a signifi cant infl uence to the response. Therefore, it is clear that the level of Mg in cells of Scenedesmus obliquus is directly related to A, B and C factors used. In conditions of relatively constant temperature and pH, this factor would control reactions related to the accumulation of Mg in the microalgae cells. The value of the R-squared statistic supports this statement because the results obtained explain 80.67% of the variability of the Mg concentration.

Analysis of the Response Surface Methodology (RSM)
shows that the highest Mg concentration was 9697.65 [mg (100g dw ) -1 ] (25 times higher than in the initial biomass), observed for the culture grown at 0.27 g L -1 of sodium nitrate, 0.00 g L -1 of sodium acetate and 95.28 μE m -2 s -1 ( Figure 6). Under these conditions the Mg concentration in Scenedesmus obliquus is higher than that previously reported by Hernández, et al. [19] for Scenedesmus almeriensis growing in pig manure (511 [mg (100g dw ) -1 ]).
An adult individual considered healthy requires 420 mg of Mg per day, value recommended by the Food and Drug Administration [20], which could be achieved with a daily consumption of 4.33 g of Scenedesmus obliquus, produced under the indicated conditions. Otherwise, a daily intake of 116 g of Scenedesmus obliquus will be necessary. In other cases, a daily intake of approximately 105g of Spirulina platensis or 122 g of Chlorella vulgaris will meet Mg requirements [18].

Effect of experimental factors on the Sodium (Na) concentration
The Na concentration varied between 5607. 20  For the Na concentration, the eff ects of the sodium nitrate concentration-irradiance interaction (factor AC) and the irradiance on its quadratic component (factor CC) were signifi cant. The eff ects of each factor and their interaction, as well as statistical signifi cance, are reported in fi gure 7.  The recommended daily intake for Na must be less than 2300 mg per day for adults [20]. Therefore, a daily intake of less 41 g of Scenedesmus obliquus, produced under the indicated conditions, will meet Na requirements for an adult. Sodium, normally found in foods in the form of Sodium Chloride (NaCl), is an essential mineral for the correct function of the human body. However, it is known that the high consumption of this mineral may trigger body disorders, as such cardiovascular disease and increased blood pressure [21].

Effect of experimental factors on the microminerals concentration: Fe, Zn and Mn
Among the micro-minerals, Fe was found in the largest amount, followed by Zn and Mn. Iron is an essential mineral nutrient which is present in many foods, taking as reference the food known worldwide as a source of iron, such as raw liver (5.60 [mg (100g dw ) -1 ]), leaves of spinach (19.23 [mg (100g dw ) -1 ]) and brindle raw beans (1860 [mg (100g dw ) -1 ]) [22,23].
Adult males, children and infants requires 10 mg of Fe per day, while for an adult female the recommended daily intakes is 15 mg of Fe per day [20]. These values could be achieved with the consumption of 0.50 g day -1 of Scenedesmus obliquus, produced under the indicated conditions. Otherwise, a daily intake of 5 g day -1 of Scenedesmus obliquus will be necessary. Analysis of the Response Surface Methodology (RSM) shows that the highest Zn concentration was 324 [mg (100g dw ) -1 ] (50 times higher than in the initial biomass), observed for the culture grown at 0.27 g L -1 of sodium nitrate, 0.00 g L -1 of sodium acetate and 56.44 μE m -2 s -1 ( Figure 10). Under these conditions the Zn concentration in Scenedesmus obliquus is higher than that previously reported by Tokusoglu and Ünal [18]  Biologically, Zn is a functional component of several metal-enzymes and metal-proteins, participates in many reactions of cellular metabolism, such as immune function, antioxidant defence and growth [6].
The recommended daily intake for Zn is 11 mg per day for adults [20]. This value could be achieved with the consumption of 3.39 g day -1 of Scenedesmus obliquus, produced under the indicated conditions. Otherwise, a daily intake of 160 g day -1 of Scenedesmus obliquus will be necessary. Whereas about 407 g day -1 of Isochrisis galbana, 917 g day -1 of Chlorella vulgaris and 440 g day -1 of Spirulina platensis fulfi ls Zn requirements [18]. Biologically, Mn is associated with the connective tissues and bones formation, carbohydrates and lipids metabolism, as well as growth and reproduction processes [24]. Rich sources of manganese include nuts (8.8 [mg (100g dw ) -1 ]), leafy green vegetables, such as spinach (0.9 [mg (100g dw ) -1 ]) and wheat germ (20 [mg (100g dw ) -1 ]) [25].
The recommended daily intake for Mn is 2.3 mg per day for adults [20]. This value could be achieved with the consumption of 5.98 g day -1 of Scenedesmus obliquus, produced under the indicated conditions. Otherwise, a daily intake of 10.40 g day -1 of Scenedesmus obliquus will be necessary. Whereas about 40.35 g day -1 of Isochrisis galbana, 109.52 g day -1 of Chlorella vulgaris and 60.52 g day -1 of Spirulina platensis fulfi ls Mn requirements [18].
Thus, Scenedesmus obliquus biomass produced under the indicated conditions can be considered as a far more representative source of the micro-minerals Fe, Zn and Mn.

CONCLUSIONS
The present work provided information regarding the infl uence of the nitrate and acetate sodium concentrations