Beruflich Dokumente
Kultur Dokumente
Received 17 February 2004; received in revised form 17 March 2005; accepted 27 September 2005
Available online 27 October 2006
Abstract
The cyanobacterium Spirulina platensis has been used by humans because of its nutritional and possibly medicinal eVects. Our study
evaluated the inXuence of temperature and nitrogen concentration in the medium on the production of biomass by this cyanobacterium
and the biomass composition in protein, lipid and phenolic compounds. We found that at 35 °C there was a negative eVect on biomass
production but a positive eVect on the production of protein, lipids and phenolics, the highest levels of these compounds being obtained in
Zarrouk’s medium containing 1.875 or 2.500 g l¡1 sodium nitrate. Higher biomass densities and productivity were obtained at 30 °C than
at 35 °C, but nitrogen concentration appeared to have no eVect on the amount of protein, lipid or phenolics, indicating that at 30 °C the
concentration of sodium nitrate in Zarrouk’s medium (2.50 g l¡1) can be reduced without loss of productivity, an important cost-saving
factor in large-scale cultivation.
© 2005 Elsevier Ltd. All rights reserved.
0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2005.09.030
1490 L.M. Colla et al. / Bioresource Technology 98 (2007) 1489–1493
Table 1
Cultivation of Spirulina platensis in media with diVerent sodium nitrate concentrations and at diVerent temperatures
Run NaNO3 (g l¡1) max (day¡1) t (h) P450 (mg l¡1 day¡1) Protein (%) Lipid (%) Phenolics (mg g¡1)
T D 30 °C
1 0.625 0.073 § 0.002 24–468 34.0 § 0.1 59.76 § 2.07 6.73 § 0.40 3.09 § 0.26
2 1.250 0.073 § 0.002 24–468 30.0 § 1.0 57.36 § 1.13 6.69 § 0.27 3.66 § 0.27
3 1.875 0.073 § 0.001 48–612 34.0 § 3.7 60.82 § 1.88 7.61 § 0.43 3.27 § 0.39
4 2.500 0.074 § 0.005 24–468 30.2 § 0.7 57.61 § 1.16 8.16 § 0.23 3.78 § 0.30
T D 35 °C
5 0.625 0.048 § 0.001 96–588 23.9 § 3.9 58.92 § 0.96 7.49 § 1.10 2.46 § 0.22
6 1.250 0.050 § 0.006 72–588 24.8 § 4.0 56.73 § 0.79 7.95 § 1.42 2.42 § 0.21
7 1.875 0.054 § 0.002 24–564 26.4 § 3.9 70.15 § 0.82 10.37 § 0.63 4.99 § 0.37
8 2.500 0.054 § 0.003 24–468 24.8 § 4.0 65.47 § 2.19 10.03 § 0.63 4.92 § 0.29
Except for t, all values show means § standard deviation.
max D maximum speciWc growth rate; t D start–end of the exponential growth phase; P450 D productivity at 450 h.
L.M. Colla et al. / Bioresource Technology 98 (2007) 1489–1493 1491
from the log phase of the growth curves by exponential sure of CO2 in the medium is higher at 30 °C than at 35 °C,
regression. Productivities was calculated from the equa- leading to a higher concentration of bicarbonate and conse-
tion P D (Xi ¡ X0)/ti, where P Dproductivity (mg l¡1 day¡1), quently an increased rate of photosynthesis. Another factor
X0 D initial biomass density (mg l¡1), Xi D biomass density that should be considered is that at higher temperatures (i.e.
at time i (mg l¡1) and ti D time interval (h) between X0 and Xi. 35 °C) there is an increase in dark cycle respiratory activity
Protein was determined by the micro-Kjeldahl method in which the cells use reserve material (e.g. carbohydrates)
according to AOAC standard methods (AOAC, 1995) in for respiration and a concomitant decrease in cell weight
which protein is assumed to contain 16% nitrogen. Four (Vonshak et al., 1982). According to Torzillo and Bernar-
replicates were analyzed for each lyophilized biomass sam- dini (1991), for outdoor cultures of Spirulina up to 34% of
ple. the biomass produced during the daylight period may be
Lipid content was evaluated using Folch’s method lost through respiration at night.
(Folch and Lees, 1957) by extracting lipids in a 2:1 chloro- Values for maximum speciWc growth rate (max), dura-
form/methanol mixture and determining lipid content tion of the exponential phase (t), productivity at 450 h
gravimetrically. Three replicates were used for each lyophi- (P450), and protein, lipid and phenolic compound content
lized biomass sample. are shown in Table 1.
Phenolic compounds were determined by extraction In general the biomass and max values were higher at
with methanol, followed by partitioning with hexane and 30 °C (0.073–0.074 day¡1) than they were at 35 °C (0.048–
precipitation of non-phenolics with Ba(OH)2 and ZnSO4. 0.054 day¡1). In Spirulina, the lower the population density,
Total phenolics were determined spectrophotometrically by the higher the speciWc growth rate. This is to be expected
the Folin–Ciocalteau method using tyrosine as standard for a system that is primarily light-limited because reducing
(Singleton and Rossi, 1965). Five replicates were used for the population density increases the availability of light to
each lyophilized biomass sample. each cell. However, the eVect of decreasing the population
density in a light-limited system is most pronounced at high
3. Results and discussion temperatures and much less so at low temperatures (Von-
shak et al., 1982). This explains why the highest max values
The growth curves for 30 °C are shown in Fig. 1a while were observed at 30 °C and not at 35 °C. The max values at
Fig. 1b shows the curves for 35 °C. Because the S. platensis 30 °C appear not to have been aVected by sodium nitrate
cells had previously been adapted to the medium there was concentration. The highest productivity (P450 D 30–
no lag phase. The biomass concentrations achieved in the 34 mg l¡1 day¡1) was obtained at 30 °C, and it thus appears
30 °C runs (0.82–0.92 g l¡1) were higher than in the 35 °C that the concentration of sodium nitrate in Zarrouk’s
runs (0.59–0.65 g l¡1). It has been shown by previous work- medium (2.500 g l¡1) could be reduced to 0.625 g l¡1 without
ers (Danesi et al., 2001; Vonshak, 1997) that the optimal loss of productivity, decrease production costs in large-
growth temperature for S. platensis is between 30 and scale cultivation.
35 °C, with 40 °C deWnitely being deleterious to this cyano- The p-values (Table 2) show that the concentration of
bacterium. In respect to increase in biomass, the best sodium nitrate in Zarrouk’s medium had a signiWcant eVect
responses were obtained at 30 °C, which agrees with the on the production of protein, lipid and phenolics, while cul-
studies by Danesi et al. (2001). tivation temperature had a signiWcant eVect on all variables.
The fact that the highest biomass values occurred at There were signiWcant interactions between protein, lipid
30 °C may have been due to the fact that the partial pres- and phenolics contents at the 99% conWdence interval,
1.00 1.00
0.80 0.80
Biomass (g/l)
Biomass (g/l)
0.60 0.60
0.40 0.40
0.20 0.20
0.00 0.00
0 200 400 600 800 0 200 400 600 800
Time (h) Time (h)
(a) (b)
Fig. 1. Biomass density growth curves of Spirulina platensis growing in media containing diVerent concentrations of sodium nitrate: (a) 30 °C (Run 1 䊉,
Run 2 䊏, Run 3 䊊, Run 4 䊐); (b) 35 °C (Run 5 䊉, Run 6 䊏, Run 7 䊊, Run 8 䊐). Sodium nitrate concentration was 0.625, 1.250, 1.875 and 2.500 g l¡1
respectively for Runs 1–4, the same concentrations being used in the same order for Runs 5–8 respectively.
1492 L.M. Colla et al. / Bioresource Technology 98 (2007) 1489–1493
ways that lead to the formation of Xavonols and phenyl- platensis in microcosms. World Journal of Microbiology and Biotech-
propanols are related to the pentose-phosphate (Calvin) nology 16, 15–18.
Danesi, E.D.G., Rangel, C.O., Pelizer, L.H., Carvalho, J.C.M., Sato, S.,
cycle, and the amounts synthesized are characteristic of Moraes, I.O., 2001. Production of Spirulina platensis under diVerent
each organism. These same pathways can lead to the for- temperatures and urea feeding regimes for chlorophyll attainment. In:
mation of such compounds in cyanobacteria because the Proceedings of the Eighth International Congress on Engineering and
Calvin cycle is part of the photosynthetic carbon Wxing Food, vol. 2, pp. 1978–1982.
mechanism of these organisms (Fay, 1993). Duval, B., Shetty, K., 2001. The stimulation of phenolics and antioxidant
activity in pea (Pisum sativum) elicited by genetically transformed anise
Although the mechanisms used by cyanobacteria to syn- root extract. Journal of Food Biochemistry 25 (5), 361–377.
thesize phenolic compounds and their metabolic functions Estrada, J.E., Bescós, P., Villar Del Fresno, A.M., 2001. Antioxidant activ-
in cells are still unknown, we found that at 35 °C there was ity of diVerent fractions of Spirulina platensis protean extract. Il Far-
a signiWcant increase in the quantity of phenolic com- maco 56, 497–500.
pounds synthesized by the cells, and this increase was also Fay, P., 1993. The Blue-Greens. Edward Arnold, London. ISBN 0-7131-
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related to an increase in other cellular components such as Folch, J., Lees, M., 1957. A simple method for isolation and puriWcation of
proteins and lipids. In spite of the fact that biomass produc- total lipids from animal tissues. Journal of Biological Chemistry 226,
tion at 30 °C presented better results in terms of productiv- 497–509.
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advantages related to the quantities of useful compounds naga, T., 1992. InXuence of ammonium chloride on growth and fatty
acid production by Spirulina platensis. Applied Biochemistry and Bio-
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Miranda, M.S., Cintra, R.G., Barros, S.B.M., Filho, J.M., 1998. Antioxi-
4. Conclusions dant activity of the microalga Spirulina maxima. Brazilian Journal of
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In this paper we have demonstrated that temperature has Olguín, E., Galicia, S., Angulo-Guerrero, O., Hernández, E., 2001. The
eVect of low light Xux and nitrogen deWciency on the chemical compo-
an important inXuence on the production of biomass, pro- sition of Spirulina sp. (Arthospira) grown on digested pig waste. Biore-
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of these compounds being obtained at 35 °C and 1.875 g l¡1 green algae under diVerent nitrogen regimes. Phytochemistry 23 (2),
207–216.
sodium nitrate or 2.500 g l¡1 sodium nitrate). Therefore, cul- Qiang, H., Guterman, H., Richmond, A., 1996. Physiological characteris-
tivation at 35 °C can be exploited when the purpose is to pro- tics of Spirulina platensis (cyanobacteria) cultured at ultrahigh cell den-
duce S. platensis with nutritional characteristics. Highest sities. Journal of Phycology 32, 1066–1073.
biomass density and productivity were obtained at 30 °C, Ramamoorthy, A., Premakumari, S., 1996. EVect of supplementation of
although nitrogen concentration appeared to have no eVect Spirulina on hypercholesterolemic patients. Journal of Food Science
and Technology 33 (2), 124–128.
on the quantity of protein, lipid or phenolics produced, Rechner, A.R., Spencer, J.P.E., Kuhnle, G., Hahn, U., Rice-Evans, C.A.,
which indicates that at this temperature the concentration of 2001. Novel biomarkers of the metabolism of caVeic acid derivatives
sodium nitrate in Zarrouk’s medium (2.50 g l¡1) can be in vivo. Free Radical Biology and Medicine 30 (11), 1213–1222.
reduced to 0.625 g l¡1 without loss of biomass productivity. Singleton, V.L., Rossi, J.A., 1965. Colorimetry of total phenolics with
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