Professional Documents
Culture Documents
Growth and Composition of Arthrospira Photobioreactor Using Ammonium Nitrate As The Nitrogen Source in A Fed-Batch Process
Growth and Composition of Arthrospira Photobioreactor Using Ammonium Nitrate As The Nitrogen Source in A Fed-Batch Process
Vol. 32, No. 02, pp. 347 - 356, April - June, 2015
dx.doi.org/10.1590/0104-6632.20150322s00003062
(Submitted: October 25, 2013 ; Revised: July 21, 2014 ; Accepted: July 22, 2014)
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. platensis in
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.
used nitrogen sources, and several studies have A 22 plus star central composite design combined
demonstrated the feasibility of replacing these con- with response surface methodology was used to opti-
ventional nitrogen sources with low-cost alternatives mize the production of A. platensis in a tubular photo-
such as urea, ammonium sulfate and ammonium bioreactor, employing ammonium nitrate as the nitro-
chloride (Carvalho et al., 2004; Bezerra et al., 2008; gen source using a fed-batch process. The analysis
Matsudo et al., 2012; Ferreira et al., 2010; Avila- measured the influence of light intensity (I) and the
Leon et al., 2012). Depending on the purpose of total amount of added ammonium nitrate (Mt) on
cultivation (e.g., pigments, carotenoid, fatty acids, growth and kinetic parameters such as maximum cell
biomass), different variables may be controlled in the concentration (Xm), cell productivity (PX), biomass
growth of A. platensis. For example, the quantity and yield on nitrogen (YX/N) and on the protein and lipid
quality of the nitrogen source have a significant ef- contents.
fect 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 MATERIAL AND METHODS
adopted in S. platensis production, ammonium salts
are in part present in the medium as ammonia, which Microorganism Used and Inoculum Preparation
is volatile and can even be toxic to the microorgan-
ism, depending on its concentration (Abeliovich & The strain Spirulina platensis UTEX 1926, recently
Azov, 1976; Muro-Pastor & Florencio, 2003; Bezerra re-classified as Arthrospira platensis (Nordstedt)
et al., 2008). Even when urea is used as a nitrogen Gomont (Silva et al., 1996), was obtained from the
source, ammonia is formed either by its hydrolysis University of Texas Culture Collection. It was grown
under alkaline conditions (Danesi et al., 2002) or by in 500 mL-Erlenmeyer flasks containing 250 mL of
urease activity (Shimamatsu, 2004). Spirulina platen- Schlösser medium (Schlösser, 1982), containing the
sis can be cultivated by batch, continuous, semi- following nutrients (g L-1): NaHCO3, 13.61; Na2CO3,
continuous (Reichert et al., 2006) and fed-batch 4.03; NaCl, 1.00; K2SO4, 1.00; NaNO3, 2.50; K2HPO4,
processes (Carvalho et al., 2004). The latter process, 0.50; MgSO4.7H2O, 0.20; CaCl2.2H2O, 0.04. All nu-
when conducted at both a suitable feeding time and trients were dissolved in distilled water containing
feeding rate to supply the nitrogen source according (per liter): 6 mL of metal solution (750 mg Na2EDTA,
to A. platensis demand, can avoid any inhibitory 97 mg FeCl3.6H2O, 41 mg MnCl2.4H2O, 5.0 mg ZnCl2,
ammonia concentration in the medium (Bezerra et 2 mg CoCl2.6H2O, 4.0 mg Na2MoO4.2H2O) and 1 mL
al., 2008; Carvalho et al., 2004; Matsudo et al., of micronutrient solution (50.0 mg Na2EDTA, 618 mg
2009). Ammonium nitrate provides the culture with a H3BO3, 19.6 mg CuSO4.5H2O, 44.0 mg ZnSO4.7H2O,
readily assimilable nitrogen source (ammonia) and 20.0 mg CoCl2.6H2O, 12.6 mg MnCl2.4H2O, 12.6 mg
one reserve nitrogen source (nitrate) because ammo- Na2MoO4.2H2O).
nia is the preferred type of nitrogen for S. platensis These flasks were maintained on a rotary shaker at
(Boussiba, 1989). Therefore, high biomass concen- 100 rpm, 30 °C and 6.0 klux (72 µmol photons m-2 s-1).
tration and productivity are expected when such a The resulting suspension was harvested during expo-
nitrogen source is employed in A. platensis cultiva- nential growth, filtered and washed twice in a physio-
tion using a fed-batch process. logical solution (0.9% NaCl) to remove absorbed
The light intensity is also an important variable in salts, including NaNO3. The cells were then resus-
cyanobacteria cultivation. High values of light inten- pended in the same Schlösser medium without any
sity promote growth parameters such as maximum nitrogen source and used to inoculate the airlift
specific growth rate, whereas low values result in a photobioreactor. The initial cell concentration was
biomass that is rich in pigments and proteins (Danesi fixed at 400 mg l-1, expressed as dry weight (Soletto
et al., 2004). et al., 2008).
The cultivation of S. platensis can be performed
in conventional open ponds or closed photobioreac- Photobioreactor
tors (Carvalho et al., 2013). The utilization of tubular
photobioreactors in the cultivation of A. platensis is All of the experiments were carried out in a tubu-
recommended for ammonia compounds (Ferreira et lar photobioreactor with an airlift system (Figure 1).
al., 2010) because nitrogen loss by off-gassing and The photobioreactor used in this study was devel-
water evaporation can be avoided, thereby increasing oped at the Fermentation Technology Laboratory of
cellular concentration and reducing the cultivation the Department of Biochemical and Pharmaceutical
cost. Technology of São Paulo University.
Brazilian Journal of Chemical Engineering
Arthrospira platensis in a Fed-Batch Process Using Ammonium Nitrate 349
Experimental Design
Table 1: Factorial design for fed-batch cultivation of A. platensis using different light intensities, concen-
trations of ammonium nitrate as a nitrogen source and related experimental results (Parts A and B).
Brazilian Journal of Chemical Engineering Vol. 32, No. 02, pp. 347 - 356, April - June, 2015
350 L. C. Cruz-Martínez, C. K. C. Jesus, M. C. Matsudo, E. D. G. Danesi, S. Sato and J. C. M. Carvalho
The addition of NH4NO3 was made according to the cultivation time (TC), which is the time at which
the method proposed by Matsudo et al. (2009) for Xm was reached:
nitrogen addition, using the following equation:
PX = ( X m – X i ) / TC (3)
M = 1.33 + k ·t (1)
Statistical Analysis
where M is the amount of NH4NO3 added until time t
(days). The initial amount of NH4NO3 was 1.33 mM,
The response surface methodology (RSM) was
and k corresponded to the quantity of NH4NO3 added
used in this study to evaluate the effects of light in-
daily. The total feeding time (tf) was 6 days. When
tensity (I) and total ammonium nitrate addition (Mt)
t = tf, the M corresponded to the total quantity of
on the fed-batch growth of A. platensis. This allowed
NH4NO3 added (Mt) (Table 1).
the determination of the influence of the two inde-
pendent variables on the three response variables
Analytical Methods
selected for this study: Xm, PX and YX/N. Multivari-
able regression analyses were performed under the
The dry cell concentration was determined by op-
conditions that had preliminarily been selected for
tical density at 560 nm using a calibration curve
the experimental design (Table 1). Such a design was
(Leduy and Therien, 1997). The dry weight of A.
based on the methodology called ‘‘star planning”
platensis biomass was determined using a digital
(Barros-Neto et al., 2003), which consists of 2 fac-
balance after filtering cells on a 0.8 µm filter and
tors in 5 levels of independent variables (runs 1–8).
drying at 60 °C overnight. A good linear relationship
The central point was repeated 3 times (runs 9–11) to
between the dry weight concentration (DWC) and
check the reproducibility of the results.
the OD560 nm was obtained. The ammonia concen-
The response equation and the corresponding sur-
tration was determined using the phenol-hypochlorite
face plot were generated in this study according to
method at 640 nm with a calibration curve (Solorzano,
the following general polynomial equation:
1969). The nitrate concentration was determined in
accordance with the methodology described by Vogel
(2002). The total carbonate concentration was deter- y = b0 + b1 X 1 + b2 X 2 + b11 X 12
mined using the titration method (Pierce and Hae- (4)
nisch, 1948). The pH was determined daily using a + b22 X 2 2 + b12 X 1 X 2
potentiometer. At the end of each cultivation, cells
were filtered, washed with distilled water and dried where y is the predicted response of the dependent
at 55 °C. The total lipids of the dried biomass were variable (Xm, PX or YX/N), X1 and X2 are the codified
extracted in a Soxhlet using a 2:1 (v/v) chloroform- values of the independent variables I and Mt, respec-
methanol mixture in accordance with the method tively, and the parameter b is the polynomial coeffi-
described by Olguín et al. (2001). The protein con- cient to be estimated by model fitting using the S-
tent of the dry biomass was determined using the PLUS 2000 program. Values < 0.10 were considered
Kjeldahl method (Association of Official Analytical significant for regression analysis, and p < 0.05 was
Chemists, 1984) with a conversion factor of 6.25. considered significant in the analyses of variance
(ANOVA).
Parameter Calculations
The biomass yield on nitrogen (YX/N) was calcu- RESULTS AND DISCUSSION
lated as the ratio of the produced cell mass to the
amount of nitrogen added to the system: Comparing NaNO3 and NH4NO3 as a Nitrogen
Source
YX/N = V ( X m – X i ) / mN (2)
The growth curves of all of the runs with NH4NO3
where Xm is the maximum cell concentration in the did not show a lag phase, as shown in Figure 2, with
reactor and Xi is the cell concentration in the inocu- the exception of run 7, in which there was no growth
lum, V is the working volume of the reactor, and mN from the first day of cultivation because of the in-
is the total mass of nitrogen added to the reactor. hibitory ammonia concentration (≥7.3 mM) (Carvalho
The cell productivity (PX) was calculated as the et al., 2004). The absence of a lag phase was ex-
ratio of the variation in cell concentration (Xm - Xi) to pected, primarily because nitrate was the nitrogen
source used by A. platensis during inoculum cultiva- ≤ 15 mM and the light intensity was in the range of
tion. The total carbonate concentration was practi- 60 µmol photons m-2 s-1 ≤ I ≤ 180 µmol photons m-2 s-1.
cally constant due to the daily pH correction using Regarding the biomass yield on nitrogen (YX/N), in
carbon dioxide, and generally remained higher than the central point runs of this study (runs 9 - 11; I =
8.0 g l-1. This procedure resulted in avoiding any lack 120 µmol photons m-2 s-1 and Mt = 15 mM), a mean
of carbon source during the cultivation (Matsudo et value of YX/N = 8.55 mg mg-1 was obtained. This
al., 2012). This control permits us to assume that the value is higher than that obtained under the same
variations obtained during the cultivation occurred cultivation conditions (tubular photobioreactor, light
due to the independent variables studied in this work. intensity, microorganism and pure CO2) but employ-
Concerning the nitrate concentration, the residual ing the original culture medium with NaNO3 as ni-
concentration of this nitrogen form was higher in trogen source (YX/N = 4.5 mg mg-1) (Bezerra et al.,
runs that were conducted with higher concentrations 2013). It is important to note that this parameter is
of ammonium nitrate, such as runs 3, 4 and 7 (Table calculated by accounting for the total amount of
1). This behavior was expected because when both nitrogen added to the system (Equation (2)) without
ammonia and nitrate are present in the medium, am- considering the residual nitrogen remaining at the
monia is preferentially assimilated (Boussiba, 1989). 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 effi-
ciency of the conversion of total nitrogen added to
cells thus greatly decreases.
Figure 2: A. platensis growth at different concentra- The use of ammoniacal nitrate is justified to ob-
tions of NH4NO3 and light intensities. (●): Run 2 (7.5 tain an increase in the biomass production of A.
mM, 180 µmol photons m-2 s-1); (∆): Run 3 (22.5 mM, platensis as a consequence of the adequate supply of
60 µmol photons m-2 s-1); (◊): Run 5 (4.4 mM, 120 nitrogen to the culture system. The constant presence
µmol photons m-2 s-1); (■): Run 7 (25.6 mM, 120 of nitrate prevents the nitrogen deficiency found in
µmol photons m-2 s-1); (▲): Run 11 (15.0 mM, 120 cultivations with ammonia, and the presence of am-
µmol photons m-2 s-1). monia, which is readily assimilated by cyanobacte-
ria, requires less nitrate and thus helps reduce the
The central point runs (runs 9 - 11; I = 120 µmol costs of the culture medium. Moreover, when the
photons m-2 s-1 and Mt = 15 mM) provided a mean cells use ammonia for growth, energy is saved be-
value of Xm = 3995.33 mg L-1 in 7 to 8 days. This Xm cause less nitrate is converted to ammonia by nitrate
value is slightly higher than the value obtained under reductase (Hatori and Myers, 1996).
the same cultivation condition (tubular photobioreac-
tor, light intensity, microorganism and pure CO2) but Optimization of A. platensis Growth Using Am-
employing the original culture medium, with NaNO3 monium Nitrate as a Nitrogen Source
as a nitrogen source (Xm = 3209 mg L-1) (Bezerra et
al., 2013). Additionally, the cultivation time was the Influence of Independent Variables on the Growth
same (7 days). These results indicate that it would be of A. platensis
feasible to replace the original nitrogen source with
NH4NO3. Nevertheless, the unsatisfactory results in Depending on the concentration of the nitrogen
the runs with the lowest (run 5, Table 1) and highest source and the light intensity evaluated in this work,
(run 7, Table 1) total amounts of ammonium nitrate different values of maximum cell concentration (Xm),
indicate a need to optimize the addition of this nutri- cell productivity (PX) and biomass yield on nitrogen
ent. In fact, higher values of Xm were obtained when (YX/N) were obtained in a tubular photobioreactor
the nitrogen supply was in the range of 7.5 mM ≤ Mt (Table 1).
Brazilian Journal of Chemical Engineering Vol. 32, No. 02, pp. 347 - 356, April - June, 2015
352 L. C. Cruz-Martínez, C. K. C. Jesus, M. C. Matsudo, E. D. G. Danesi, S. Sato and J. C. M. Carvalho
Brazilian Journal of Chemical Engineering Vol. 32, No. 02, pp. 347 - 356, April - June, 2015
354 L. C. Cruz-Martínez, C. K. C. Jesus, M. C. Matsudo, E. D. G. Danesi, S. Sato and J. C. M. Carvalho
CONCLUSIONS
YX/N Yield of biomass on nitrogen (mg·mg-1) Carvalho, J. C. M., Francisco, F. R., Almeida, K. A.,
V Reactor volume (L) Sato, S., Converti, A., Cultivation of Arthrospira
TC Cultivation time (days) (Spirulina) platensis (Cyanophyceae) by fed-batch
X1 Codified values of I (dimensionless) addition of ammonium chloride at exponentially
X2 Codified values of Mt (dimensionless) increasing feeding rates. Journal of Phycology, 40,
p. 589-597 (2004).
Cohen, Z., The Chemicals of Spirulina. In: A.
REFERENCES Vonshak, Ed. Spirulina platensis (Arthrospira):
Physiology, Cell-Biology and Biotechnology,
Abeliovich, A., Azov, Y., Toxicity of ammonia to Taylor and Francis, London (1997).
algae in sewage oxidation ponds. Appl. Environm. Danesi, E. D. G., Rangel-Yagui, C. O., Carvalho, J. C.
Microbiol., 31, p. 801-806 (1976). M., Sato, S., An investigation of effect of replac-
Association of Official Analytical Chemists, Official ing nitrate by urea in the growth and production
Methods of Analysis of the Association of Official of chlorophyll by Spirulina platensis. Biomass
Analytical Chemists. 14th (Ed.), AOAC, Arlington Bioenerg., 23, p. 261-269 (2002).
(1984). Danesi, E. D. G., Rangel-Yagui, C. O., Carvalho, J. C.
Avila-Leon, I., Matsudo, M. C., Sato, S., & Carvalho, M., Sato, S., Effect of reducing the light intensity
J. C. M., Arthrospira platensis biomass with high on the growth and production of chlorophyll by
protein content cultivated in continuous process Spirulina platensis. Biomass and Bioenergy, 26, p.
using urea as nitrogen source. Journal of Applied 329-335 (2004).
Microbiology, 112(6), pp. 1086-94 (2012). Faintuch, B., Análise comparativa da produção de
Barros-Neto, B., Scarminio, I. S., & Bruns, R. E., biomassa a partir de três cianobactérias empre-
Como Fazer Experimentos. Editora da UNICAMP, gando distintas fontes nitrogenadas. Master Dis-
Campinas, p. 401 (2003). (In Portuguese). sertation. University of São Paulo (1989). (In
Belay, A., Culture of Spirulina Outdoors - the Portuguese).
Earthrise Farms Experience. In: A. Vonshak (Ed.), Ferreira, L. S., Rodrigues, M. S., Converti, A., Sato,
Spirulina platensis (Arthrospira): Physiology, Cell- S., Carvalho, J. C. M., A new approach to ammo-
Biology and Biotechnology, Taylor and Francis, nium sulphate feeding for fed-batch Arthrospira
London (1997). (Spirulina) platensis cultivation in tubular photo-
Bezerra, R. P., Matsudo, M. C., Converti, A., Sato, S., bioreactor. Biotechnol. Progress, 26, p. 1271-1277
Carvalho, J. C. M., Influence of ammonium chlo- (2010).
ride feeding time and light intensity on the culti- Fratelli, F., Siquini, T., Prado, S., Higashi, H., Converti,
vation of Spirulina (Arthrospira) platensis. Bio- A., Carvalho, J., Effect of medium composition
technology and Bioengineering, 100, p. 297-305 on the production of tetanus toxin by Clostridium
(2008). tetani. Biotechnol. Progr., 21, p. 756-761 (2005).
Bezerra, R. P., Matsudo, M. C., Sato, S., Converti, A., Hatori, A., Myers, J., Reduction of nitrate and nitrite
& Carvalho, J. C. M., Fed-batch cultivation of Ar- by subcellular preparations of Anabaena cylin-
throspira platensis using carbon dioxide from drica. Plant Physiol. Biochem., 41, p. 1031-1036
alcoholic fermentation and urea as carbon and (1996).
nitrogen sources. BioEnergy Research, 6, p. 1118- Leduy, A., Therien, N., An improved method for op-
1125 (2013). tical density measurement of the semimicroscopic
Boussiba, S., Ammonia uptake in the alkalophilic blue algae Spirulina maxima. Biotechnol. Bioeng.,
cyanobacterium Spirulina platensis. Plant Cell 19, p. 1219-1224 (1977).
Physiol., 30, p. 303-308 (1989). Matsudo, M., Bezerra, R., Sato, S., Perego, P.,
Carlozzi, P., Pinzani, E., Growth characteristics of Converti, A., Carvalho, J., Repeated fed-batch
Arthrospira platensis cultured inside a new closed- cultivation of Arthrospira (Spirulina) platensis
coil photobioreactor incorporating a mandrel to using urea as nitrogen source. Biochemical En-
control culture temperature. Biotechnol. Bioeng., gineering Journal, 43, p. 52-57 (2009).
90, p. 675-684 (2005). Matsudo, M. C., Bezerra, R. P., Sato, S., Converti, A.,
Carvalho, J. C. M., Bezerra, R. P., Matsudo, M. C., Carvalho, J. C. M., Photosynthetic efficiency and
Sato, S., Cultivation of Arthrospira (Spirulina) rate of CO2 assimilation by Arthrospira (Spirulina)
platensis by Fed-Batch Process. In: J. Lee (Ed.), platensis continuously cultivated in a tubular
Advanced Biofuels and Bioproducts, Springer photobioreactor. Biotechnology Journal, 7, p.
New York, New York (2013). 1412-7 (2012).
Brazilian Journal of Chemical Engineering Vol. 32, No. 02, pp. 347 - 356, April - June, 2015
356 L. C. Cruz-Martínez, C. K. C. Jesus, M. C. Matsudo, E. D. G. Danesi, S. Sato and J. C. M. Carvalho
Moraes, C. C., Sala, L., Cerveira, G. P., Kalil, S. J., benthic marine algae of the Indian Ocean. B.
C-phycocyanin extraction from Spirulina platensis University of California Publications in Botany,
wet biomass. Brazilian Journal of Chemical Engi- Berkeley (1996).
neering, 28, p. 45-49 (2011). Soletto, D., Binaghi, L., Ferrari, L., Lodi, A.,
Muro-Pastor, M., Florencio, F., Regulation of ammo- Carvalho, J. C. M., Zilli, M., Converti, A., Effects
nium assimilation in cyanobacteria. Plant Physiol. of carbon dioxide feeding rate and light intensity
Bioch., 41, p. 595-603 (2003). on the fed-batch pulse-feeding cultivation of
Olguín, E., Galicia, S., Hernández, E., Angulo, O., Spirulina platensis in helical photobioreactor.
The effect of low light flux and nitrogen defi- Biochemical Engineering Journal, 39, p. 369-375
ciency on the chemical composition of Spirulina (2008).
sp. growth on pig waste. Bioresour. Technol., 77, Solorzano, L., Determination of ammonia in natural
p. 19-24 (2001). waters by the phenol hypochloride method. Limnol.
Pierce, W. C., Haenisch, E. L., Quantitative Analysis. Oceanogr., 14, p. 799-801 (1969).
3rd (Ed.), John Wiley & Sons, Inc, New York Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert,
(1948). A., Commercial applications of microalgae. J. Biosci.
Piorreck, M., Baasch, K., Pohl, P., Biomass produc- Bioeng., 101, p. 87-96 (2006).
tion, total protein, chlorophylls, lipids and fatty Taragano, V., Pilosof, A., Application of Doehlert
acids of freshwater green and blue-green algae designs for water activity, pH, and fermentation
under different nitrogen regimes. Phytochemistry, time optimization for Aspergillus niger, pecti-
23, p. 207-216 (1984). nolytic activities production in solid-state and
Reichert, C. D. C., Reinehr, C. O., Costa, J. A. V., submerged fermentation. Enzyme Microb. Tech.,
Semicontinuous cultivation of the cyanobacte- 25, p. 411-419 (1999).
rium Spirulina platensis in a closed photobioreac- Viswanathan, P., Kulkarni, P., Full factorial design to
tor. Brazilian Journal of Chemical Engineering, 23, study fermentative production of inulinase using
p. 23-28 (2006). inulin from Kuth (Saussurea lappa) root power
Sanchez-Luna, L. D., Bezerra, R. P., Matsudo, M. C., by Aspergillus niger Van Teighem UV11 mutant.
Sato, S., Converti, A., Carvalho, J. C. M., Influ- Bioresour. Technol., p. 54, 117-121 (1995).
ence of pH, temperature, and urea molar flowrate Vogel, A., Análise Química Quantitativa. 6th (Ed.),
on arthrospira platensis fed-batch cultivation: A Livros Tecnicos e Cientificos, Rio de Janeiro
kinetic and thermodynamic approach. Biotechnol. (2002). (In Portuguese).
and Bioeng., 96, p. 702-711 (2007). Vonshak, A., Spirulina: Growth, Physiology and Bio-
Sassano, C. E. N., Gioielli, L. A., Ferreira, L. S., chemistry. In: A. Vonshak (Ed.), Spirulina platensis
Rodrigues, M. S., Sato, S., Converti, A., Carvalho, (Arthrospira): Physiology, Cell-Biology and Bio-
J. C. M., Evaluation of the composition of con- technology. Taylor and Francis, London (1997).
tinuously-cultivated Arthrospira (Spirulina) plat- Vonshak, A., Appendices. In: A. Vonshak, Ed. Spirulina
ensis using ammonium chloride as nitrogen source. platensis (Arthrospira): Physiology, Cell Biology
Biomass and Bioenergy, 34, p. 1732-1738 (2010). and Biotechnology. Taylor and Francis, London
Schlösser, U. G., Berichte der Deutschen Botanischen (1997b).
Gesellschaft, 95, p. 181-276 (1982). (In German). Vonshak, A., Cheung, S. M., Chen, F., Mixotrophic
Shimamatsu, H., Mass production of Spirulina, an edi- growth modifies the response of Spirulina (Ar-
ble microalga. Hydrobiologia, 512, p. 39-44 (2004). throspira) platensis (Cyanobacteria) cells to light.
Silva, P., Basson, P., Moe, R., Catalogue of the J. Phycol., 36, p. 675-679 (2000).