Jurnal Kultur Danik

Journal of Agricultural Science and Technology A 2 (2012) 824-832 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250

Effect of N and P on the Uptake of Magnesium and Iron and on the Production of Carotenoids and Chlorophyll

by the Microalgae Nannochloropsis sp.

Telma Encarnao1, Hugh D. Burrows1, Alberto C. Pais1, Maria G. Campos2 and Adrien Kremer3

1..Department of Chemistry, Faculty of Science and Technology, University of Coimbra, Rua Larga, Coimbra 3004-535, Portugal

2. Faculty of Pharmacy, University of Coimbra, Rua do Norte, Coimbra 3000-295, Portugal

3. Faculty of Pharmacy, University of Angers, Angers 49045, France

Received: January 16, 2012 / Published: June 20, 2012. Abstract: This study investigate the effect of the concentration of nitrate and phosphate, present in the culture medium, on the chemical and biochemical composition of the products from the marine microalgae, Nannochloropsis sp.. Experimental design allowed the assessment, in a systematic way, of the response of the microalgae to the nitrate and phosphate concentrations, and the way they lead to changes in the total amount of carotenoids, chlorophyll a, iron and magnesium produced or uptaken. The total carotenoids presented a higher yield when cultivated under lower phosphate concentrations, but showed no change with nitrate concentration. Chlorophyll a yield increased in the presence of higher concentrations of nitrogen and lower concentrations of phosphorus. There was an increase in the amount of iron absorbed by cells when higher levels of nitrates were present, but the effect is insignificant with phosphates. The magnesium content was not significantly affected by culture manipulation. The results also showed that the biomass yield of the microalgae Nannochloropsis sp. was negatively affected by the N/P ratio. The antioxidative potential of the microalgae, in contrast, was found to increase with the N/P ratio. Key words: Antioxidants, biomass, DPPH, experimental design, redfield ratio. 1. Introduction

Microalgae are the source of biosynthesis of many valuable compounds, including pigments, antioxidants, and dietary supplements. These microorganisms can synthesize toxins, and produce a wide range of bioactive molecules with antibiotic, anti-cancer, anti-inflammatory and antiviral and other properties [1]. Moreover, microalgae also have great potential for removing nitrates and phosphates from wastewaters and in reducing the greenhouse effect if used in air treatment plants for the removal of CO2, NOx and SOx from air streams and industrial flue gases [2]. The growth of microalgae is dependent on an adequate supply of essential macronutrients

Corresponding author: Telma Encarnao, M.Sc., research fields: microalgae biotechnology and bioactive chemistry. E-mail: [email protected].

(carbon, nitrogen, phosphorus), metal ions (sodium, potassium and calcium), together with micronutrients, such as traces of other metal ions (iron, zinc, copper). Metals ions, such as iron and magnesium, play an important role in photosynthesis. In particular, magnesium occupies a strategic position as the central element of the chlorophyll molecule, and all microalgal species have an absolute need for this element. Magnesium is also involved in the aggregation of ribosomes in functional units and in the formation of catalase [3]. Iron is the most important limiting nutrient of all the micronutrient metals present in the oceans [4] and is important in many metabolic functions of phytoplankton, such as electron transport in the Calvin cycle, the respiratory electron transport processes, nitrate and nitrite reductions, nitrogen fixation, the synthesis of chlorophyll, and

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Effect of N and P on the Uptake of Magnesium and Iron and on the Production of Carotenoids and Chlorophyll by the Microalgae Nannochloropsis sp.

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detoxification/degradation of reactive oxygen species (such as superoxide radicals and hydrogen peroxide). It is also implicated in a number of other biosynthetic pathways and degradation reactions [5]. Apart from nutrient availability, various factors, including light, pH, temperature and salinity, affect not only photosynthesis and biomass productivity, but also activity and mechanisms of cellular metabolism. Within the different components present in the culture media, the amount of carbon, nitrogen, phosphorus and sulfur can influence growth and biochemical composition of microalgal cells, leading to effects on the growth rate, the levels of proteins, lipids and pigments, and also to changes in biological activity [6]. As a consequence, it is anticipated that manipulation of culture conditions can be exploited to favour formation of a product with value added nutritional or other properties such as antioxidant activity [7]. Increased levels of reactive oxygen species are generally believed to produce oxidative damage to macromolecules such as lipids, nucleic acids and proteins. Microalgae have developed a variety of defense mechanisms that include antioxidant ones, which are attributed to a wide range of species, including carotenoids, fatty acids and enzymes, such as catalase, glutathione peroxidase and superoxide dismutase [8-10]. Recently, there has been an increasing interest in marine organism in the search for new antioxidants, which may help in reducing or preventing free radical-induced tissue injury. Although many plant species have been investigated for their antioxidant activity, little is known regarding marine organism [11]. The marine species used in this research, Nannochloropsis sp., is a unicellular microalga that has been used in aquaculture as a feedstock for marine fish larvae and crustaceans. This species is well known as a source of a variety of products, including polyunsaturated fatty acids (including eicosapentaenoic acid), pigments (carotenoids and chlorophyll), minerals (Na, K, Ca, Mg, Fe, Cu, Zn), vitamins and proteins [12]. What is

needed is a way of optimizing this production. Experimental design has been used in many

different applications [13], although its application to studies concerning biosynthesis has been relatively limited. This is a serious omission, since it can provide a systematic assessment of the impact of variables under control that is likely to be extremely valuable in the directed production of nutrients or fine chemicals using microalgae. The results obtained with this technique provide an approximate function for the response based on controlled variables. It is possible to assess the importance of each variable, and also the degree of interaction between them. It also indicates the way each variable affects the response. In one of the simplest approaches, the experiments are carried out in two levels per variable, resulting in 2k different experiments, for k factors. This is known as the 2k full factorial design, and will be the approach used in the present work. In this study, the effect and the response of marine microalgae Nannochloropsis sp. to concentrations of nitrates and phosphates have been assessed, in a novel approach, using an experimental design. The antioxidant activity was also determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method [14] and related to the N/P ratio. The biomass yield from the microalgae Nannochloropsis sp. was also correlated with the N/P ratio.

2. Material and Methods

2.1 Microorganism Source and Culture Conditions

Nannochloropsis sp. was obtained from Florida Aqua Farms, Inc., USA. The culture is supplied in disk containing ca. 3 billions cells. One disk is cultivated for 6 days in 2 L commercial media Cell-hi F2P (Varicon Aqua Solution, Malvern, UK), based upon the f/2 medium [15]. The temperature was regulated in a water bath at 23 1 C under partial illumination (with a 18:6 photoperiod) using fluorescent lamps at an irradiance level of 36.5 mol m-2s-1. This setup was used as inoculum. To test the


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effects of nutrients, 10 equal fractions of 200 mL of inoculum were subsequently transferred to sterilized 1.5 L polyethylene terephthalate bottles, to which were added 800 mL of culture medium Cell-hi TEViT (Varicon Aqua Solution, Malvern, UK), based upon the f/2 medium [15] deprived of nitrates and phosphates. Nitrate levels were set at 0.003 M NO3- (low N) and 0.006 M NO3- (high N) and phosphate levels were set at 0.0003 M PO43- (low P) and 0.001 M PO43- (high P). The cultures were kept for 14 days in the water bath (23 1 C), under the above conditions of partial illumination. All culture studies were carried out in duplicate and all media were previously sterilized by microwave irradiation.

2.2 Biomass Production

After cultivation, the biomass was decanted and filtered through Nylon 66 membrane filters with a pore diameter of 0.2 m, and washed with a solution of ammonium bicarbonate followed by ultrapure water, using a method adapted from Zhu and Lee [16] to remove NaCl and remnants of the culture medium. The biomass was then frozen at -20 C in test tubes and dried in a Labcomco, Freeze Dry System/ Freezone 4.5 apparatus.

2.3 Iron and Magnesium Quantification

Quantification of iron and magnesium in cultured cells was performed by atomic emission spectrometry using inductively coupled plasma (ICP Unicam 701 Series Systems). The lyophilized samples were subjected to digestion by microwave irradiation to destroy the organic matrix of the biological samples. Amounts of 10 to 20 mg of lyophilized biomass were weighed into the digesters, to which were added 5 mL of nitric acid (65%), 2 mL hydrogen peroxide (30% w/v, 100 vol) and 5 mL of ultrapure water (Milipore subsequently filtered through nylon membrane filters 66 with a pore size of 0.2 m in diameter). A standard was prepared with 10 mL ultrapure water, 5 mL nitric acid, 2 mL of hydrogen peroxide and 1 mL of solution

icm 2.0 (2.0 mg/L Fe) or 0.5 mL sol. Int. Ctrl II (100 mg/L Mg) and 10 mL of ultrapure water, and a blank with just the corresponding amounts of ultrapure water, nitric acid and hydrogen peroxide. Solution icm 2.0 was prepared by dilution of a multielement solution from VHG-LABS. Int Ctrl II solution was prepared by dilution of the magnesium stock solution from Merck. The samples were placed in a microwave digester (Milestone MLS Digestion Unit 1200 Mega) for 30 minutes, with the temperature of samples increased to 180 5 C within about 5 minutes and kept at this temperature for a further 10 minutes.

2.4 Chlorophyll a and Total Carotenoids Quantification

For the quantification of chlorophyll a and total carotenoids, 18 mg of dry biomass was weighed, to which 10 mL of methanol was added. The mixture was shaken vigorously by vortexing for 30 seconds. Subsequently, the samples were centrifuged at 4,000 rpm for 10 min. UV-visible spectral measurements were made using the absorbance of the supernatant with the solvent as reference, using a Shimadzu UV-2450 spectrophotometer. The absorbance data were converted into concentrations, using the Equations described by Porra [17], Lichtenthaler and Wellburn [18].

2.5 Antioxidant Capacity Assessment

The antioxidant capacity was evaluated using the method of free radical capture by 2,2-diphenyl-1-picrylhydrazyl (DPPH) as described by Campos et al. [14]; 5.96 mg of DPPH were weighed and dissolved in 250 mL of ethanol during 30 min and the solution was subjected to ultrasounds. This solution was freshly prepared daily and was kept in the dark when not being used.

Samples of 10 mg of lyophilized biomass were extracted with 3 mL ethanol. The mixture was homogenized by vortexing and subjected to ultrasounds for 1 h in an ice bath. The extracts were


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then centrifuged and the supernatant was used in the determination of the free radical scavenging properties. Different amounts of extracts were added to 2 mL of DPPH solution, and the mixtures were homogenized by vortexing. These mixtures rested for 10 minutes to allow complete reaction, following which the absorbance of the DPPH radical remaining in each sample was determined at 517 nm. This analytical wavelength () was chosen to avoid interferences from microalgae pigments. Blanks were prepared in a similar way and measured under identical conditions. The above sequence of procedures was repeated using methanol. Differences were observed between results in the two solvents. While we do not have an explanation for these differences at present, this is in agreement with the work of Sharma and Bhat [19] and may reflect specific solvent effects. For this discussion, the results in ethanol will be used.

2.6 Experimental Design

A full two-level factorial 2k was carried out to determine the effect of the concentrations of nitrates and phosphates on iron, magnesium, chlorophyll a and total carotenoids yields of microalgal cells, and establish the degree of interaction between the factors. These concentrations were expressed in coded values, -1 and +1 for the lower and upper values, respectively.

The factor analysis used 22 experiments in different conditions (-1, -1), (-1, +1), (+1, -1) and (+1, +1), with two repetitions for each set of conditions yielding a total of 8 runs, and provide a function of four parameters [13].

R = a0 + a1x1 + a2x2 + a12x1x2 (1) where R is the response of the system and x1 and x2 are the values of the factors, which are the nitrate and phosphate concentrations, respectively. Expressions such as Equation (1) provide a summary of the behavior of the response in terms of the controlled variables, and are very useful even when only two factors are considered. The parameter a0 represents the average of all the experiments, which would correspond to the response in the absence of any

influence stemming from the factors. The parameters a1 and a2 correspond to what is often called the main effects of factors x1 and x2. They are the first measure of the influence of these factors, but the influence of each one may be changed by the value at which the other one is operated. This change is taken into consideration by the interaction term, in which parameter a12 is present. A thorough discussion of this term has been given by Vitorino et al. [20]. It is noted that the interaction term is often overlooked in designs in which an individual variable is examined, with all others frozen.

3. Results and Discussion

3.1 Biomass Production

The results presented in Table 1 show that biomass production is affected by nitrate and phosphate concentrations. The results of experimental design for biomass production, using Equation (1), are presented in Table 2. As expected, the data confirm the direct influence of nitrates and phosphates on the production of biomass, as can be seen by the relatively high level of confidence of the respective coefficients.

The increase of algal biomass with phosphates is consistent with the results of Lai et al. [21]. In contrast, Hu and Gao [22], in studies at a lower range of P concentrations, found a decrease of biomass yield with an increase in phosphate concentration, while Tubea et al. [23] reported a negligible effect of phosphates on the algal growth.

With respect to the influence of nitrates on the biomass production, the negative value of the coefficient indicates that, within the range of concentration values, the biomass decrease with nitrate concentration. Hu and Gao [22] also made similar observations with similar levels of nitrates. However, Ault et al. [24] and Pietilainen and Niinioja [25] observed that nitrate levels produced no significant effect on algal growth. Note, however, that these studies were conducted with different microalgae.


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Table 1 Iron, magnesium, total carotenoids, chlorophyll a, biomass and N/P ratio obtained in the Nannochloropsis sp. study after 14 days of culture. NaNO3 (M)

KH2PO4 (M)

Fe (mg/g)

Mg (mg/g)

Total carotenoids (g/g)

Chlorophyll a (g/g)

Biomass dry weight (g/L) N/P

0.006 0.0003 3.37 77.3 455.7 612.1 0.082 20 0.006 0.0003 4.75 67.1 369.2 635.5 0.063 20 0.006 0.001 3.86 78.1 290.8 435.6 0.085 6 0.006 0.001 3.81 98.3 191.8 217.6 0.093 6 0.003 0.0003 2.83 82.0 407.0 540.4 0.096 10 0.003 0.0003 2.91 93.5 351.6 445.2 0.097 10 0.003 0.001 3.46 74.8 212.1 264.6 0.11 3 0.003 0.001 2.41 74.4 289.6 368.2 0.12 3 Table 2 Values of coefficients, probability for students test using the quantities of biomass dry weight, iron, magnesium, chlorophyll a and total carotenoids are shown in Table 1. The coefficients were obtained using coded values for the controled variables, concentration of phosphates and nitrates. The lower levels of the concentrations are assigned the value -1 and the higher +1. Coefficient a0 AN AP ANP

Biomass Value 0.09 -0.01 0.009 0.0005 Probability/% > 99.9 98.8 96.2 13.0

Iron Value 3.4 0.5 -0.04 -0.07 Probability/% > 99.9 92.6 13.9 24.3

Magnesium Value 80.7 0.5 0.7 -7.3 Probability/% > 99.9 11.5 16.7 91.7

Chlorophyll a Value 439.9 35.3 -118.4 -30.2 Probability/% > 99.9 66.1 97.8 59.4

Total Carotenoids Value 321.0 5.9 -74.9 -10.7 Probability/% > 99.9 21.4 97.9 37.3

The observation of the stoichiometry of the oceans led Redfield [26] to an optimum N/P ratio of 16:1. This is known as the Redfield ratio and widely used for phytoplankton. However, it is been recognized that Redfield stoichiometry is not always related to a rapidly growing phytoplankton [27] and the ratio of nitrogen to phosphorous demands varies with growth conditions [28]. The results shown in Table 2 indicate that the interaction between nitrates and phosphates in algal biomass production is negligible. Fig. 1 is compatible with this result, indicating that the biomass does not monotonically increase with the N/P ratio. However, it seems that lower N/P ratios do result in larger amounts of biomass. The results also suggest that phosphorous could be the limiting nutrient. Previous studies [29, 28] have demonstrated that the non-Redfield ratios, to values below 10, are related with phytoplankton blooms. Encarnao [7] showed

the same results in cultures of Nannochloropsis, cultivated in the same conditions but supplemented with CO2, biomass increased with the decrease of N/P ratios. Klausmeier et al. [29] stated that different species varied in their N/P ratio, and that the Redfield stoichiometry represented an average for specific organisms. In this context, it is possible to state that a higher biomass yield of the microalgae Nannochloropsis sp. can be obtained with N/P ratio bellow the Redfield stoichiometry.

3.2 Iron and Magnesium Quantification

The iron content in cells varied with the nitrate concentration over the range under study, but remained almost independent of phosphate concentration, as can be seen from Table 1. Negligible changes in magnesium concentration were observed upon variation in nitrate or phosphate concentrations.


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Fig. 1 Biomass productivity of Nannochloropsis sp., after 14 days of culture, as function of N/P ratio. Error bars correspond to one standard deviation.

In the Table 1, the values are summarized of the coefficients obtained for Equation (1), in relation to the iron content, and respective significance. From this analysis, we can conclude that, within the range of concentration values studied, nitrate has some impact upon the presence of iron. This is seen through the high value of the corresponding coefficient, and also by the respective level of confidence, which is, however, below 95%. The positive value of this coefficient indicates that an increase in the controlled variable produces an increase in the amount of iron absorbed by the cells. With respect to the concentration of phosphate, the effect is insignificant, as is its interaction with the nitrate in the medium.

Using a similar analysis for magnesium, the results indicate that direct effects of the concentrations used as manipulated variables are extremely small. There may, however, be some hints of an interaction, which causes a nontrivial change in the amount of magnesium upon varying the concentrations of nitrate and phosphate.

It has been shown in several studies that iron plays a key role in phytoplankton growth [30, 31]. The Fe-S ferredoxin protein is an electron donor involved in the reduction of SO42- and NO3- ions. The assimilation of nitrate-nitrogen by autotrophic organisms requires the complementary presence of active enzymes that digest

the nitrogen, an energy supply in the form of ATP, reducing agents (NADPH and ferredoxin) and also a carbon source. For the incorporation of nitrogen, and reduction of nitrate NO3-, the metalloenzymes nitrate reductase and nitrite reductase need the donation of electrons from ferredoxin [32]. In this study, an increase in NO3- concentration in the medium caused a higher iron uptake by the cells, also resulting in nitrogen absorption, probably related to the synthesis of the reducing agent, ferredoxin. However, this assimilation was not accompanied by any increase in biomass production.

3.3 Chlorophyll a and Total Carotenoids Quantification

The results presented in Table 1 show that the amount of chlorophyll a increased with increasing nitrate concentration, and also with a decrease in the concentration of phosphates. Total carotenoids increased with decreasing concentrations of phosphate. However, apparently they were not affected by the concentration of nitrates.

The experimental design analysis was again applied to the system, considering now the amount of chlorophyll a as response. From careful examination, it appears that the effect of varying the concentration of phosphate is important for the chlorophyll a content


830

in the cell, as suggested by the coefficient for this concentration, and confirmed by the confidence level. The effects of nitrate concentration and the interaction between the two concentrations are relatively low.

Applying the same analysis to the variation of total carotenoids, presented in the Table 1, it can be seen from values of coefficients and their confidence levels that the concentration of phosphate is the most significant parameter. Variations of nitrate concentration or of interaction between the two concentrations do not appear to be significant.

It should be noted, however, that the mechanisms responsible for the synthesis of carotenoids are poorly understood [33].

3.4 Antioxidant Capacity Assessment

Screening of the potential antioxidant activity was carried out using a free radical scavenging assay that revealed EC50 value of ca. 300 g/mL for methanolic extracts, and around 1,000 g/mL for ethanolic extracts. Standards, such as the important free radical scavenger, ascorbic acid, showed values of 2.41 0.003 g/mL under the same conditions. Since the blanks were performed, the interference of solvent in the process can be discarded. The difference between the two solvents results could be due to extraction of different compounds. This aspect requires more investigation.

Spectroscopic measurements on the DPPH disappearance in the presence of increasing Nannochloropsis extract concentrations are shown in Figs. 2 and 3.

These results show a clear correlation between the extract concentration and antioxidative potential, which is not influenced by variations in nitrate or phosphate concentrations. A variation in the values of EC50 would normally be expected with the manipulation of the medium, due to variations in the yield of the known antioxidants, the carotenoids. A possible explanation is that the enzymes and unsaturated fatty acids also display antioxidant effects, and this may influence the values, which cannot be discriminated from the effect of carotenoids. However, attempts were made to correlate the antioxidant activity with the nitrate to phosphate ratio, rather than the individual concentrations. In Fig. 3, an obvious effect is demonstrated the increasing free radical scavenging properties with the N/P ratio. It is worth noting that the free radical scavenging properties appear to plateau at high N/P ratios, indicating that it should be possible to optimize antioxidant production with this ratio. It is of interest to recall also, that the chlorophyll a content increases with the N/P ratio. There may be an effect of photosynthetic efficiency upon the antioxidant activity.

Fig. 2 Dependence of DPPH concentration on the Nannochloropsis extract concentrations. Error bars correspond to one standard deviation.


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Fig. 3 Response of antioxidant activity of ethanolic extracts of Nannochloropsis sp. as function of N/P ratio. Error bars correspond to one standard deviation.

4. Conclusions

Although microalgae systems have many interrelated factors which are difficult to control, the application of experimental design provides a very useful tool for the interpretation and prediction in these complex systems. This allows the optimization of procedures to prepare a variety of products, and allows us to focus upon optimization of compounds yields of specific interest. Our results show that the non-Redfield stoichiometry and low ratio N/P, favor a higher biomass productivity in Nannochloropsis sp. and provides an insight into routes that allow the production of a biomass with more antioxidant activities. These results clearly demonstrate the possibility of manipulating the photosynthesis by the marine microalgae Nannochloropsis to produce important metabolites, with added value for the food industry or other applications.

Acknowledgments

The authors acknowledge Prof. A. M. dA. Rocha Gonalves and Dr. Teresa Morgado from Associao para a Inovao Tecnolgica e Qualidade (AEMITEQ) and the contribution and assistance of Drs. Lusa Ramos and Simone Ferreira in the NMR and atomic emission spectrometry experiments, respectively.

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