SciELO - Scientific Electronic Library Online

 
vol.32 issue2A MULTISTAGE GRADUAL NITROGENREDUCTION STRATEGY FOR INCREASED LIPID PRODUCTIVITY AND NITROGEN REMOVAL IN WASTEWATER USING Chlorella vulgaris AND Scenedesmus obliquusOPTIMIZATION OF THE OPERATING CONDITIONS FOR RHAMNOLIPID PRODUCTION USING SLAUGHTERHOUSE-GENERATED INDUSTRIAL FLOAT AS SUBSTRATE author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Brazilian Journal of Chemical Engineering

Print version ISSN 0104-6632On-line version ISSN 0104-6632

Braz. J. Chem. Eng. vol.32 no.2 São Paulo Apr./June 2015

http://dx.doi.org/10.1590/0104-6632.20150322s00003062 

BIOPROCESS ENGINEERING

GROWTH AND COMPOSITION OF Arthrospira (Spirulina) platensis IN A TUBULAR PHOTOBIOREACTOR USING AMMONIUM NITRATE AS THE NITROGEN SOURCE IN A FED-BATCH PROCESS

C. Cruz-Martínez1 

C. K. C. Jesus1 

M. C. Matsudo2 

E. D. G. Danesi3 

S. Sato1 

J. C. M. Carvalho1  * 

1Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo - SP, Brazil. Phone: + (55) (11) 30913886, Fax: + (55) (11) 38156386 E-mail: jcmdcarv@usp.br; jcmdcarvalho@gmail.com

2Federal University of Itajubá, Itajubá - MG, Brazil.

3Department of Food Engineering, Ponta Grossa State University, Ponta Grossa - PR, Brazil.

Abstract

NH4NO3 simultaneously provides a readily assimilable nitrogen source (ammonia) and a reserve of nitrogen (nitrate), allowing for an increase in Arthrospira platensis biomass production while reducing the cost of the cultivation medium. In this study, a 22 plus star central composite experimental design combined with response surface methodology was employed to analyze the influence of light intensity (I) and the total amount of added NH4NO3 (Mt) on a bench-scale tubular photobioreactor for fed-batch cultures. The maximum cell concentration (Xm), cell productivity (PX) and biomass yield on nitrogen (YX/N) were evaluated, as were the protein and lipid contents. Under optimized conditions (I = 148 μmol·photons·m-2·s-1 and Mt = 9.7 mM NH4NO3), Xm = 4710 ±34.4 mg·L-1, PX = 478.9 ±3.8 mg·L-1·d-1 and YX/N = 15.87 ±0.13 mg·mg-1 were obtained. The best conditions for protein content in the biomass (63.2%) were not the same as those that maximized cell growth (I = 180 μmol·photons·m-2·s-1 and Mt = 22.5 mM NH4NO3). Based on these results, it is possible to conclude that ammonium nitrate is an interesting alternate nitrogen source for the cultivation of A. platensisin a fed-batch process and could be used for other photosynthetic microorganisms.

Keywords Arthrospira (Spirulina) platensis; Fed-batch cultivation; Ammonium nitrate; Light intensity; Tubular photobioreactor

INTRODUCTION

The use of microorganisms in food production and in the environmental field, including the biofixation of CO2 and bioremediation, is a major focus of recent biotechnological studies. In this context, photosynthetic microorganisms such as cyanobacteria are of particular interest because they generate several compounds with diverse applications (Belay, 1997; Spolaore et al., 2006; Vonshak, 1997) in addition to food protein. A. platensis is an important source of cellular protein, essential fatty acids, phycocyanin and chlorophyll a (Belay, 1997; Spolaore et al., 2006, Moraes et al., 2011).

In conventional cyanobacteria cultivation, nitrate salts (sodium and potassium nitrates) are the most used nitrogen sources, and several studies have demonstrated the feasibility of replacing these conventional nitrogen sources with low-cost alternatives such as urea, ammonium sulfate and ammonium chloride (Carvalho et al., 2004; Bezerra et al., 2008; Matsudo et al., 2012; Ferreira et al., 2010; Avila-Leon et al., 2012). Depending on the purpose of cultivation (e.g., pigments, carotenoid, fatty acids, biomass), different variables may be controlled in the growth of A. platensis. For example, the quantity and quality of the nitrogen source have a significant effect on the biomass yield and quality (Piorreck et al., 1984; Danesi et al., 2002; Avila-Leon et al., 2012). Under alkaline conditions, such as those usually adopted in S. platensis production, ammonium salts are in part present in the medium as ammonia, which is volatile and can even be toxic to the microorganism, depending on its concentration (Abeliovich & Azov, 1976; Muro-Pastor & Florencio, 2003; Bezerra et al., 2008). Even when urea is used as a nitrogen source, ammonia is formed either by its hydrolysis under alkaline conditions (Danesi et al., 2002) or by urease activity (Shimamatsu, 2004). Spirulina platensis can be cultivated by batch, continuous, semicontinuous (Reichert et al., 2006) and fed-batch processes (Carvalho et al., 2004). The latter process, when conducted at both a suitable feeding time and feeding rate to supply the nitrogen source according to A. platensis demand, can avoid any inhibitory ammonia concentration in the medium (Bezerra et al., 2008; Carvalho et al., 2004; Matsudo et al., 2009). Ammonium nitrate provides the culture with a readily assimilable nitrogen source (ammonia) and one reserve nitrogen source (nitrate) because ammonia is the preferred type of nitrogen for S. platensis (Boussiba, 1989). Therefore, high biomass concentration and productivity are expected when such a nitrogen source is employed in A. platensiscultivation using a fed-batch process.

The light intensity is also an important variable in cyanobacteria cultivation. High values of light intensity promote growth parameters such as maximum specific growth rate, whereas low values result in a biomass that is rich in pigments and proteins (Danesi et al., 2004).

The cultivation of S. platensis can be performed in conventional open ponds or closed photobioreactors (Carvalho et al., 2013). The utilization of tubular photobioreactors in the cultivation of A. platensis is recommended for ammonia compounds (Ferreira et al., 2010) because nitrogen loss by off-gassing and water evaporation can be avoided, thereby increasing cellular concentration and reducing the cultivation cost.

A 22 plus star central composite design combined with response surface methodology was used to optimize the production of A. platensis in a tubular photobioreactor, employing ammonium nitrate as the nitrogen source using a fed-batch process. The analysis measured the influence of light intensity (I) and the total amount of added ammonium nitrate (Mt) on growth and kinetic parameters such as maximum cell concentration (Xm), cell productivity (PX), biomass yield on nitrogen (YX/N) and on the protein and lipid contents.

MATERIAL AND METHODS

Microorganism Used and Inoculum Preparation

The strain Spirulina platensis UTEX 1926, recently re-classified as Arthrospira platensis (Nordstedt) Gomont (Silva et al ., 1996), was obtained from the University of Texas Culture Collection. It was grown in 500 mL-Erlenmeyer flasks containing 250 mL of Schlösser medium (Schlösser, 1982), containing the following nutrients (g L-1): NaHCO3, 13.61; Na2CO3, 4.03; NaCl, 1.00; K2SO4, 1.00; NaNO3, 2.50; K2HPO4, 0.50; MgSO4.7H2O, 0.20; CaCl2.2H2O, 0.04. All nutrients were dissolved in distilled water containing (per liter): 6 mL of metal solution (750 mg Na2EDTA, 97 mg FeCl3.6H2O, 41 mg MnCl2.4H2O, 5.0 mg ZnCl2, 2 mg CoCl2.6H2O, 4.0 mg Na2MoO4.2H2O) and 1 mL of micronutrient solution (50.0 mg Na2EDTA, 618 mg H3BO3, 19.6 mg CuSO4.5H2O, 44.0 mg ZnSO4.7H2O, 20.0 mg CoCl2.6H2O, 12.6 mg MnCl2.4H2O, 12.6 mg Na2MoO4.2H2O).

These flasks were maintained on a rotary shaker at 100 rpm, 30 °C and 6.0 klux (72 μmol photons m-2 s-1). The resulting suspension was harvested during exponential growth, filtered and washed twice in a physiological solution (0.9% NaCl) to remove absorbed salts, including NaNO3. The cells were then resuspended in the same Schlösser medium without any nitrogen source and used to inoculate the airlift photobioreactor. The initial cell concentration was fixed at 400 mg l-1, expressed as dry weight (Soletto et al., 2008).

Photobioreactor

All of the experiments were carried out in a tubular photobioreactor with an airlift system (Figure 1). The photobioreactor used in this study was developed at the Fermentation Technology Laboratory of the Department of Biochemical and Pharmaceutical Technology of São Paulo University.

Figure 1 Tubular photobioreactor. (1): Degasser; (2): Condenser tube; (3): Air pump; (4): 20 W Fluorescent lamps; (5): Airlift system (Carvalho et al., 2013). 

It is made of a metal structure surrounded by 40 glass tubes that are linked by silicone rubber tubes. The glass tubes (0.01 m internal diameter (Carlozzi and Pizani, 2005), 0.015 m wall thickness and 0.5 m length) was positioned with a 2% (1.15 °) incline. At the bottom of the reactor is a "Y" tube that receives air and cell culture, leading the latter to a degasser vessel that is located at the top of the structure. This flask has a glass tube that contributes to reducing the loss of both water and ammonia by evaporation. The working volume was 3.4 L. The illuminated volume (in the tubes illuminated by the fluorescent lamps) corresponds to approximately 57% of the total working volume. The culture flow was 40 L h-1, the temperature was set at 29 ± 1 °C, and the pH was 9.5 ± 0.5 (Sanchez-Luna et al., 2007), controlled by the addition of CO2 from a cylinder (Matsudo et al., 2012).

Experimental Design

In fed-batch runs using NH4NO3, the Schlösser medium (Schlösser, 1982) lacking its original nitrogen source (NaNO3) was used, employing a feeding time of 6 days for adding NH4NO3. The total amount of nitrogen source (added twice a day) and the light intensities were adjusted according to the experimental design (Table 1).

Table 1 Factorial design for fed-batch cultivation of A. platensis using different light intensities, concentrations of ammonium nitrate as a nitrogen source and related experimental results (Parts A and B). 

Test X1a X2b Ic Mtd TCe Xmf PXg YX/Nh NH3i NO3j Lip k Ptn l
Part A. Tests of the initial experimental design
1 -1 -1 60 7.5 8 3914 439.2 16.73 0.55 * 8.3 23.8
2 +1 -1 180 7.5 10 4834 443.4 21.11 0.02 0.4 5.9 14.8
3 -1 + 1 60 22.5 5 1029 125.8 1.00 11.10 12.2 ** **
4 +1 + 1 180 22.5 5 960 112.1 0.89 10.4 10.6 17.3 63.2
5 0 -1.414 120 4.4 7 3422 431.7 24.53 0.01 0.5 3.3 12.3
6 -1.414 0 35 15.0 7 2164 252.0 4.20 0.09 9.2 12.5 41.7
7 0 + 1.414 120 25.6 4 676 69.1 0.38 10.2 12.2 ** **
8 + 1.414 0 205 15.0 8 3926 440.7 8.39 0.44 0.63 8.1 25.8
9 0 0 120 15.0 8 4194 474.2 9.03 0.10 12.4 7.6 25.5
10 0 0 120 15.0 7 3854 493.4 8.22 0.10 11.3 7.0 22.5
11 0 0 120 15.0 8 3938 442.2 8.42 0.06 7.5 9.9 29.3
Part B. Confirmation tests
12 0.47 - 0.71 148 9.7 9 4699 477.7 15.83 0.07 0.5 10.3 21.4
13 0.47 - 0.71 148 9.7 9 4683 475.9 15.77 0.04 2.6 9.4 18.9
14 0.47 - 0.71 148 9.7 9 4749 483.2 16.01 0.03 1.2 10.5 19.8

aX1 = Codified values for light intensity,

bX2 = Codified values for addition of ammonium nitrate (Mt),

cI = Light intensity values (μmol photons m-2s-1),

dMt = total amount of ammonium nitrate added (mM),

eTC = Cultivation time (days),

fXm = Maximum cell concentration (mg L-1),

gPX = Cell productivity (mg L-1d-1),

hYX/N = Yield of biomass on nitrogen (mg mg-1),

iNH3 = Final concentration of total ammonia (mM),

jNO3 = final concentration of nitrate (mM),

kLip = lipid biomass content (%),

lPtn = protein biomass content (%),

*value below the detection limit of the analytical method,

**It was not possible to quantify (cell death).

The addition of NH4NO3 was made according to the method proposed by Matsudo et al. (2009) for nitrogen addition, using the following equation:

where M is the amount of NH4NO3 added until time t (days). The initial amount of NH4NO3 was 1.33 mM, and k corresponded to the quantity of NH4NO3 added daily. The total feeding time (tf) was 6 days. When t = tf, the M corresponded to the total quantity of NH4NO3 added (Mt) (Table 1).

Analytical Methods

The dry cell concentration was determined by optical density at 560 nm using a calibration curve (Leduy and Therien, 1997). The dry weight of A. platensis biomass was determined using a digital balance after filtering cells on a 0.8 μm filter and drying at 60 °C overnight. A good linear relationship between the dry weight concentration (DWC) and the OD560 nm was obtained. The ammonia concentration was determined using the phenol-hypochlorite method at 640 nm with a calibration curve (Solorzano, 1969). The nitrate concentration was determined in accordance with the methodology described by Vogel (2002). The total carbonate concentration was determined using the titration method (Pierce and Haenisch, 1948). The pH was determined daily using a potentiometer. At the end of each cultivation, cells were filtered, washed with distilled water and dried at 55 °C.The total lipids of the dried biomass were extracted in a Soxhlet using a 2:1 (v/v) chloroform-methanol mixture in accordance with the method described by Olguín et al. (2001). The protein content of the dry biomass was determined using the Kjeldahl method (Association of Official Analytical Chemists, 1984) with a conversion factor of 6.25.

Parameter Calculations

The biomass yield on nitrogen (YX/N) was calculated as the ratio of the produced cell mass to the amount of nitrogen added to the system:

where Xm is the maximum cell concentration in the reactor and Xi is the cell concentration in the inoculum, V is the working volume of the reactor, and mN is the total mass of nitrogen added to the reactor.

The cell productivity (PX) was calculated as the ratio of the variation in cell concentration (Xm-Xi) to the cultivation time (TC), which is the time at which Xm was reached:

Statistical Analysis

The response surface methodology (RSM) was used in this study to evaluate the effects of light intensity (I) and total ammonium nitrate addition (Mt) on the fed-batch growth of A. platensis. This allowed the determination of the influence of the two independent variables on the three response variables selected for this study: Xm, PX and YX/N. Multivariable regression analyses were performed under the conditions that had preliminarily been selected for the experimental design (Table 1). Such a design was based on the methodology called "star planning" (Barros-Neto et al., 2003), which consists of 2 factors in 5 levels of independent variables (runs 1-8). The central point was repeated 3 times (runs 9-11) to check the reproducibility of the results.

The response equation and the corresponding surface plot were generated in this study according to the following general polynomial equation:

where y is the predicted response of the dependent variable (Xm, PX or YX/N), X1 and X2 are the codified values of the independent variables I and Mt, respectively, and the parameter b is the polynomial coefficient to be estimated by model fitting using the SPLUS 2000 program. Values < 0.10 were considered significant for regression analysis, and p < 0.05 was considered significant in the analyses of variance (ANOVA).

RESULTS AND DISCUSSIONS

Comparing NaNO3 and NH4NO3 as a Nitrogen Source

The growth curves of all of the runs with NH4NO3 did not show a lag phase, as shown in Figure 2, with the exception of run 7, in which there was no growth from the first day of cultivation because of the inhibitory ammonia concentration (≥7.3 mM) (Carvalho et al., 2004). The absence of a lag phase was expected, primarily because nitrate was the nitrogen source used by A. platensis during inoculum cultivation. The total carbonate concentration was practically constant due to the daily pH correction using carbon dioxide, and generally remained higher than 8.0 g l-1. This procedure resulted in avoiding any lack of carbon source during the cultivation (Matsudo et al., 2012). This control permits us to assume that the variations obtained during the cultivation occurred due to the independent variables studied in this work. Concerning the nitrate concentration, the residual concentration of this nitrogen form was higher in runs that were conducted with higher concentrations of ammonium nitrate, such as runs 3, 4 and 7 (Table 1). This behavior was expected because when both ammonia and nitrate are present in the medium, ammonia is preferentially assimilated (Boussiba, 1989).

Figure 2  A. platensis growth at different concentrations of NH4NO3 and light intensities. (●): Run 2 (7.5 mM, 180 μmol photons m-2 s-1); (∆): Run 3 (22.5 mM, 60 ?mol photons m-2s-1); (◊): Run 5 (4.4 mM, 120 μmol photons m-2 s-1); (■): Run 7 (25.6 mM, 120 μmol photons m-2 s-1); (▲): Run 11 (15.0 mM, 120 μmol photons m-2 s-1). 

The central point runs (runs 9 - 11; I = 120μmol photons m-2 s-1 and Mt = 15 mM) provided a mean value of Xm = 3995.33 mg L-1 in 7 to 8 days. This Xm value is slightly higher than the value obtained under the same cultivation condition (tubular photobioreactor, light intensity, microorganism and pure CO2) but employing the original culture medium, with NaNO3 as a nitrogen source (Xm = 3209 mg L-1) (Bezerra et al., 2013). Additionally, the cultivation time was the same (7 days). These results indicate that it would be feasible to replace the original nitrogen source with NH4NO3. Nevertheless, the unsatisfactory results in the runs with the lowest (run 5, Table 1) and highest (run 7, Table 1) total amounts of ammonium nitrate indicate a need to optimize the addition of this nutrient. In fact, higher values of Xm were obtained when the nitrogen supply was in the range of 7.5 mM ≤ Mt ≤ 15 mM and the light intensity was in the range of 60 μmol photons m-2 s-1 ≤I ≤ 180 μmol photons m-2 s-1.

Regarding the biomass yield on nitrogen (YX/N), in the central point runs of this study (runs 9 - 11; I = 120 μmol photons m-2 s-1 and Mt = 15 mM), a mean value of YX/N = 8.55 mg mg-1 was obtained. This value is higher than that obtained under the same cultivation conditions (tubular photobioreactor, light intensity, microorganism and pure CO2) but employing the original culture medium with NaNO3 as nitrogen source (YX/N = 4.5 mg mg-1) (Bezerra et al., 2013). It is important to note that this parameter is calculated by accounting for the total amount of nitrogen added to the system (Equation (2)) without considering the residual nitrogen remaining at the end of the cultivation.

When using NH4NO3 as a nitrogen source, the concentration of ammonia inside the reactor must be maintained at low levels (less than 1.6 mM) due to the well-known inhibitory effect that ammonia can exert on A. platensis growth (Carvalho et al., 2004). As a consequence, practically all of the nitrogen added during cell growth is converted to biomass. Conversely, when using only nitrate as the nitrogen source, higher concentrations (greater than 10 mM) must be maintained in the culture medium to prevent growth limitation (Faintuch, 1989), and the efficiency of the conversion of total nitrogen added to cells thus greatly decreases.

The use of ammoniacal nitrate is justified to obtain an increase in the biomass production of A. platensis as a consequence of the adequate supply of nitrogen to the culture system. The constant presence of nitrate prevents the nitrogen deficiency found in cultivations with ammonia, and the presence of ammonia, which is readily assimilated by cyanobacteria, requires less nitrate and thus helps reduce the costs of the culture medium. Moreover, when the cells use ammonia for growth, energy is saved because less nitrate is converted to ammonia by nitrate reductase (Hatori and Myers, 1996).

Optimization of A. platensis Growth Using Ammonium Nitrate as a Nitrogen Source

Influence of Independent Variables on the Growth of A. platensis

Depending on the concentration of the nitrogen source and the light intensity evaluated in this work, different values of maximum cell concentration (Xm), cell productivity (PX) and biomass yield on nitrogen (YX/N) were obtained in a tubular photobioreactor (Table 1).

Because different reactor conditions lead to distinct operational conditions, the relationship between Mt and I must be evaluated simultaneously to optimize A. platensis growth.

As shown in Table 1, when the total amount of ammonium nitrate added (Mt) was equal to or greater than 22.5 mM (runs 3, 4 and 7), the resulting Xm, PX and YX/N were low, with Xm reaching 676 mg l-1 when Mt = 25.6 mM (run 7), irrespective of the light intensity employed. This fact likely occurred because of the increasing ammonia concentration during cell growth, reaching 10 mM by the end of the cultivation (Table 1), a concentration that is considered to inhibit the growth of cyanobacteria (Abeliovich and Azov, 1976; Carvalho et al., 2004). In fact, such high values of ammonia concentrations led to cell death in runs 3 and 7. In run 4, despite high concentrations of ammonia during cultivation (22.5 mM), cell death was not observed, likely because of the high light intensity employed (180 μmol photons m-2 s-1). The best Xm results were obtained using 7.5 mM ≤ Mt ≤ 15 mM (runs 1, 2, 8 and central runs). In fact, Xm = 4834 mg l-1 was obtained in run 2, which was performed with Mt = 7.5 mM NH4NO3and I = 180 μmol photons m-2s-1. Although the lowest addition of ammonium nitrate (4.4 mM) led to a decrease in Xm (3422 mg l-1; run 5, Table 1), this is not a very considerable decrease compared with the Xm obtained when the highest Mt value was used (Xm = 676 mg l-1; run 7, Table 1). These findings suggest that cell growth is more strongly influenced by high ammonia concentrations than it is by low ammonia concentrations.

A clear difference was also observed in the results of Xm for the same value of light intensity (e.g., runs 1 and 3, Table 1), thus demonstrating that the main parameter that influences cell growth is the total amount of ammonium nitrate added. Conversely, as stressed above, the light intensity may be an important variable for attenuating ammonium toxicity because a higher light intensity (up to a saturating point) results in greater cell growth and, consequently, a higher assimilation of ammonia, which rapidly reduces the concentration of this nitrogen source (run 4).

The achievement of the stationary phase either at the lower (≤ 60 μmol photons m-2 s-1) or higher (≥ 180 μmol photons m-2 s-1) light intensities, even associated with the suitable nitrogen concentrations, could have been the consequence of the shading effect (Vonshak et al., 2000).

The analysis of multiple regression applied to the Xm data (Table 1, Part A) showed that this parameter was a satisfactory function of both codified values of I and Mt, as represented in Equation (5):

The adjusted model (Equation (5)) generated an adjusted determination coefficient (R2) of 0.87, i.e., it explained 87% of the variability in the maximum cell concentration. This value is considered satisfactory for our purposes and is comparable to the values typically reported in the literature for bioprocesses (0.8) (Viswanathan and Kulkarni, 1995; Taragano and Pilosof, 1999; Fratelli et al., 2005). Moreover, the analysis of variance of the regression revealed that it is statistically significant (p < 0.002). The corresponding response surface is shown in Figure 3. The value of Xm, as estimated by the model at the optimal point (4565 mg L-1) corresponds to X1 = 0.47 and X2 = -0.71, which are both inside the planned experimental range, as indicated in Figure 3. Such an optimal value of Xm was only 2.9% smaller than the experimental value obtained in the confirmation runs (4710 ± 34.4 mg l-1, Table 1, part B), thus confirming the suitability of the model. In fact, the comparison between the values calculated by the model and the experimental values of Xm showed a satisfactory relationship, thus validating the model for estimating the optimal conditions for the biomass production of A. platensis using ammonium nitrate as a nitrogen source.

Figure 3 Response surface for maximum cell concentration (Xm) as a function of the codified values of light intensity (X1) and the total amount of ammonium nitrate added (X2). The arrow indicates the optimized Xm

Cell productivity (PX) as a function of the independent variables presented a behavior similar to that found for maximum cell concentration, which can be ascribed to the low difference in the cultivation time (TC), both in runs with low cell growth (runs 3, 4 and 7, Table 1) and in runs with relatively high cell growth (runs 1, 2, 8-11, Table 1). PX was shown to be a quadratic function of X1 and X2 (Figure 4), with negative values of the corresponding quadratic coefficients, as shown in Equation (6):

Figure 4 Response surface for cell productivity (PX) as a function of the codified values of light intensity (X1) and the total amount of ammonium nitrate added (X2). 

From the data shown in Figure 4, it is possible to infer that x1 (light intensity) had less influence on PX than x2 (total amount of ammonium nitrate) did.

The equation obtained in the regression analysis to describe the biomass yield on nitrogen was as follows:

Similar to Xm and PX, YX/N was a quadratic function of both x1 and x2. As shown in Figure 5, it is possible to infer that x1 (light intensity) had less influence on YX/N than x2(total amount of ammonium nitrate) did. Nevertheless, it is worth noting that only in this variable did the interaction effect between independent variables occur (b12 = -1.12). Figure 5 shows that higher YX/N values were obtained in the runs carried out at low values of Mt, which is in agreement with the results obtained by Carvalho et al. (2004), who cultivated A. platensis in open ponds using ammonium chloride as the nitrogen source.

Figure 5 Response surface for biomass yield on nitrogen (YX/N) as a function of the codified values of light intensity (X1) and the total amount of ammonium nitrate added (X2). 

Influence of Independent Variables on the Protein and Lipid Contents

The highest content of proteins in the biomass was obtained in run 4 (63.2%), in which the values of Xm (960 mg L-1) and PX (112.1 mg L-1d-1) were lower due to the high total amount of NH4NO3 added to the reactor (22.5 mM NH4NO3). Despite the higher Xm obtained in run 2, the protein content in the biomass composition was very low (14.8%; Table 1). In fact, in run 2, the lower protein content indicated that there was not enough nitrogen for its accumulation in the form of protein. Thus, the cyanobacterium grew and accumulated energy, most likely in the form of carbohydrates (Sassano et al., 2010).

Regarding the regression of protein content in the biomass, only values of X2 ≤ 0 were considered because X2 > 0 led to cell death in most runs. Equation (8) represents such a regression:

It is interesting to note that, in contrast with the Xm and YX/N values, the highest protein values were obtained when high levels of total ammonium nitrate were employed in cell cultivation (Figure 6). The protein content in runs 12-14 showed an average value of 20.0 ± 1.3%, while the average value of the lipid content in the biomass was 10.1 ± 0.6%, similar to the values observed in runs 9 to 11 (25.8 ± 3.4% for protein content and 8.2 ± 1.5% for lipid content).

Figure 6 Response surface for biomass protein content (Ptn) as a function of the codified values of light intensity (X1) and the total amount of ammonium nitrate added (X2). The arrow indicates the highest protein value. 

These protein content values are lower than expected for A. platensis (50 - 60%), according to Vonshak (1997b). Bezerra et al. (2008), who studied the effect of light intensity and ammonium chloride feeding time on the cultivation of A. platensis, achieved an average protein content of 35.9% in cultivations conducted under optimized conditions for that independent variable. Increased levels of nitrogen may favor protein accumulation in the biomass. In this study, the addition of optimized amounts of nitrogen that were sufficient for cell growth is likely to be one of the reasons for the reduced protein content, in contrast with the results from Matsudo et al. (2012), who obtained a protein content as high as 56% and employed urea as a nitrogen source under a continuous process.

Conversely, by calculating the protein productivity (i.e., multiplying the values of cell productivity and the fraction corresponding to protein in the biomass), one can conclude that, even with a lower protein content, the overall protein productivity was much higher in the central point runs (runs 9-11, PPTN = 120.5 ± 9.2 mg L-1d-1) than it was in run 4 with the highest protein content (70.8 mg L-1d-1).

No effect of the light intensity on the biomass lipid content was detected. However, this variable was a positive linear function of the total ammonium nitrate addition (Mt). The maximum value of this dependent variable (17.3%) was obtained in run 4, where Mt = 22.5 mM. Similarly, the lowest lipid content was obtained in run 5 (4.4 mM NH4NO3), with a value of 3.3% (Table 1), due to the lack of nitrogen. Evaluating different levels of nitrogen sources in the cultivation of A. platensis, Piorreck et al. (1984) observed that the lipid content decreased under conditions of nitrogen limitation. The lipid content obtained in most of the runs is in the range expected for this photosynthetic microorganism. According to Cohen (1997), cyanobacteria are generally poor in lipids, commonly containing 6 - 13%, half of which are fatty acids.

CONCLUSIONS

This study demonstrated that light intensity (I) and the total amount of added ammonium nitrate (Mt) influence the growth of A. platensis. Values of I = 148 μmol photons m-2 s-1 and Mt = 9.7 mM NH4NO3 were the best combination for obtaining high values of Xm, with an average value of 4710 ± 34.4 mg L-1. Such a value is very close to that estimated by the model for the optimal point (4565 mg L-1). Under such conditions, the mean values of PX and YX/N were 478.9 ± 3.8 mg L-1 d-1 and 15.87 ± 0.13, respectively. It was possible to confirm the suitability of the multivariable regression analysis as a tool for obtaining optimized experimental conditions for Xm using ammonium nitrate as a nitrogen source. The best conditions found for cell growth were not the same as those for maximizing protein content. However, considering the overall protein productivity, the optimized conditions for cell growth were also appropriate for producing high-quality single cell protein. Our results show that ammonium nitrate is an interesting alternative nitrogen source for the cultivation of A. platensis in a fed-batch process and that it can be used for other photosynthetic microorganisms.

NOMENCLATURE

ACKNOWLEDGEMENTS

We acknowledge the financial support from “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP), Brazil (Grant 09/53265-6) and “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES), Brazil. We also acknowledge Laís de Lara Capurro Guimarães for help with the experiments.

REFERENCES

Abeliovich, A., Azov, Y., Toxicity of ammonia to algae in sewage oxidation ponds. Appl. Environm. Microbiol., 31, p. 801-806 (1976). [ Links ]

Association of Official Analytical Chemists, Official Methods of Analysis of the Association of Official Analytical Chemists. 14th (Ed.), AOAC, Arlington (1984). [ Links ]

Avila-Leon, I., Matsudo, M. C., Sato, S., & Carvalho, J. C. M.,Arthrospira platensis biomass with high protein content cultivated in continuous process using urea as nitrogen source. Journal of Applied Microbiology, 112(6), pp. 1086-94 (2012). [ Links ]

Barros-Neto, B., Scarminio, I. S., & Bruns, R. E., Como Fazer Experimentos. Editora da UNICAMP, Campinas, p. 401 (2003). (In Portuguese). [ Links ]

Belay, A., Culture ofSpirulina Outdoors -the Earthrise Farms Experience. In: A. Vonshak (Ed.), Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology, Taylor and Francis, London (1997). [ Links ]

Bezerra, R. P., Matsudo, M. C., Converti, A., Sato, S., Carvalho, J. C. M., Influence of ammonium chloride feeding time and light intensity on the cultivation ofSpirulina (Arthrospira) platensis. Biotechnology and Bioengineering, 100, p. 297-305 (2008). [ Links ]

Bezerra, R. P., Matsudo, M. C., Sato, S., Converti, A., & Carvalho, J. C. M., Fed-batch cultivation ofArthrospira platensis using carbon dioxide from alcoholic fermentation and urea as carbon and nitrogen sources. BioEnergy Research, 6, p. 1118-1125 (2013). [ Links ]

Boussiba, S., Ammonia uptake in the alkalophilic cyanobacteriumSpirulina platensis. Plant Cell Physiol., 30, p. 303-308 (1989). [ Links ]

Carlozzi, P., Pinzani, E., Growth characteristics ofArthrospira platensis cultured inside a new closedcoil photobioreactor incorporating a mandrel to control culture temperature. Biotechnol. Bioeng., 90, p. 675-684 (2005). [ Links ]

Carvalho, J. C. M., Bezerra, R. P., Matsudo, M. C., Sato, S., Cultivation ofArthrospira (Spirulina) platensis by Fed-Batch Process. In: J. Lee (Ed.), Advanced Biofuels and Bioproducts, Springer New York, New York (2013). [ Links ]

Carvalho, J. C. M., Francisco, F. R., Almeida, K. A., Sato, S., Converti, A., Cultivation ofArthrospira (Spirulina) platensis(Cyanophyceae) by fed-batch addition of ammonium chloride at exponentially increasing feeding rates. Journal of Phycology, 40 p. 589-597 (2004). [ Links ]

Cohen, Z., The Chemicals of Spirulina. In: A. Vonshak, Ed.Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology, Taylor and Francis, London (1997). [ Links ]

Danesi, E. D. G., Rangel-Yagui, C. O., Carvalho, J. C. M., Sato, S., An investigation of effect of replacing nitrate by urea in the growth and production of chlorophyll bySpirulina platensis. Biomass Bioenerg., 23, p. 261-269 (2002). [ Links ]

Danesi, E. D. G., Rangel-Yagui, C. O., Carvalho, J. C. M., Sato, S., Effect of reducing the light intensity on the growth and production of chlorophyll bySpirulina platensis. Biomass and Bioenergy, 26, p. 329-335 (2004). [ Links ]

Faintuch, B., Análise comparativa da produção de biomassa a partir de três cianobactérias empregando distintas fontes nitrogenadas. Master Dissertation. University of São Paulo (1989). (In Portuguese). [ Links ]

Ferreira, L. S., Rodrigues, M. S., Converti, A., Sato, S., Carvalho, J. C. M., A new approach to ammonium sulphate feeding for fed-batchArthrospira (Spirulina) platensis cultivation in tubular photobioreactor. Biotechnol. Progress, 26, p. 1271-1277 (2010). [ Links ]

Fratelli, F., Siquini, T., Prado, S., Higashi, H., Converti, A., Carvalho, J., Effect of medium composition on the production of tetanus toxin byClostridium tetani. Biotechnol. Progr., 21, p. 756-761 (2005). [ Links ]

Hatori, A., Myers, J., Reduction of nitrate and nitrite by subcellular preparations ofAnabaena cylindrica. Plant Physiol. Biochem., 41, p. 1031-1036 (1996). [ Links ]

Leduy, A., Therien, N., An improved method for optical density measurement of the semimicroscopic blue algaeSpirulina maxima. Biotechnol. Bioeng., 19, p. 1219-1224 (1977). [ Links ]

Matsudo, M., Bezerra, R., Sato, S., Perego, P., Converti, A., Carvalho, J., Repeated fed-batch cultivation ofArthrospira (Spirulina) platensis using urea as nitrogen source. Biochemical Engineering Journal, 43, p. 52-57 (2009). [ Links ]

Matsudo, M. C., Bezerra, R. P., Sato, S., Converti, A., Carvalho, J. C. M., Photosynthetic efficiency and rate of CO2 assimilation by Arthrospira (Spirulina) platensis continuously cultivated in a tubular photobioreactor. Biotechnology Journal, 7, p. 1412-7 (2012). [ Links ]

Moraes, C. C., Sala, L., Cerveira, G. P., Kalil, S. J., C-phycocyanin extraction fromSpirulina platensis wet biomass. Brazilian Journal of Chemical Engineering, 28, p. 45-49 (2011). [ Links ]

Muro-Pastor, M., Florencio, F., Regulation of ammonium assimilation in cyanobacteria. Plant Physiol. Bioch., 41, p. 595-603 (2003). [ Links ]

Olguín, E., Galicia, S., Hernández, E., Angulo, O., The effect of low light flux and nitrogen deficiency on the chemical composition of Spirulina sp. growth on pig waste. Bioresour. Technol., 77, p. 19-24 (2001). [ Links ]

Pierce, W. C., Haenisch, E. L., Quantitative Analysis. 3rd (Ed.), John Wiley & Sons, Inc, New York (1948). [ Links ]

Piorreck, M., Baasch, K., Pohl, P., Biomass production, total protein, chlorophylls, lipids and fatty acids of freshwater green and blue-green algae under different nitrogen regimes. Phytochemistry, 23, p. 207-216 (1984). [ Links ]

Reichert, C. D. C., Reinehr, C. O., Costa, J. A. V., Semicontinuous cultivation of the cyanobacteriumSpirulina platensis in a closed photobioreactor. Brazilian Journal of Chemical Engineering, 23, p. 23-28 (2006). [ Links ]

Sanchez-Luna, L. D., Bezerra, R. P., Matsudo, M. C., Sato, S., Converti, A., Carvalho, J. C. M., Influence of pH, temperature, and urea molar flowrate on arthrospira platensis fed-batch cultivation: A kinetic and thermodynamic approach. Biotechnol. and Bioeng., 96, p. 702-711 (2007). [ Links ]

Sassano, C. E. N., Gioielli, L. A., Ferreira, L. S., Rodrigues, M. S., Sato, S., Converti, A., Carvalho, J. C. M., Evaluation of the composition of continuously-cultivatedArthrospira (Spirulina) platensis using ammonium chloride as nitrogen source. Biomass and Bioenergy, 34, p. 1732-1738 (2010). [ Links ]

Schlösser, U. G., Berichte der Deutschen Botanischen Gesellschaft, 95, p. 181-276 (1982). (In German) [ Links ]

Shimamatsu, H., Mass production ofSpirulina, an edible microalga. Hydrobiologia, 512, p. 39-44 (2004). [ Links ]

Silva, P., Basson, P., Moe, R., Catalogue of the benthic marine algae of the Indian Ocean. B. University of California Publications in Botany, Berkeley (1996). [ Links ]

Soletto, D., Binaghi, L., Ferrari, L., Lodi, A., Carvalho, J. C. M., Zilli, M., Converti, A., Effects of carbon dioxide feeding rate and light intensity on the fed-batch pulse-feeding cultivation ofSpirulina platensis in helical photobioreactor. Biochemical Engineering Journal, 39, p. 369-375 (2008). [ Links ]

Solorzano, L., Determination of ammonia in natural waters by the phenol hypochloride method. Limnol. Oceanogr., 14, p. 799-801 (1969). [ Links ]

Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A., Commercial applications of microalgae. J. Biosci. Bioeng., 101, p. 87-96 (2006). [ Links ]

Taragano, V., Pilosof, A., Application of Doehlert designs for water activity, pH, and fermentation time optimization forAspergillus niger, pectinolytic activities production in solid-state and submerged fermentation. Enzyme Microb. Tech., 25, p. 411-419 (1999). [ Links ]

Viswanathan, P., Kulkarni, P., Full factorial design to study fermentative production of inulinase using inulin from Kuth (Saussurea lappa) root power by Aspergillus niger Van Teighem UV11 mutant. Bioresour. Technol., p. 54, 117-121 (1995). [ Links ]

Vogel, A., Análise Química Quantitativa. 6th (Ed.), Livros Tecnicos e Cientificos, Rio de Janeiro (2002). (In Portuguese). [ Links ]

Vonshak, A.,Spirulina: Growth, Physiology and Biochemistry. In: A. Vonshak (Ed.), Spirulina platensis (Arthrospira): Physiology, Cell-Biology and Biotechnology. Taylor and Francis, London (1997). [ Links ]

Vonshak, A., Appendices. In: A. Vonshak, Ed.Spirulina platensis (Arthrospira): Physiology, Cell Biology and Biotechnology. Taylor and Francis, London (1997b). [ Links ]

Vonshak, A., Cheung, S. M., Chen, F., Mixotrophic growth modifies the response ofSpirulina (Arthrospira) platensis(Cyanobacteria) cells to light. J. Phycol., 36, p. 675-679 (2000). [ Links ]

Received: October 25, 2013; Revised: July 21, 2014; Accepted: July 22, 2014

* To whom correspondence should be addressed

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited.