Growth and chemical composition of Spirulina maximaand Spirulina platensis biomass at differenttemperatures

OLIVEIRA, M.A.C.L. DE*, MONTEIRO, M.P.C., ROBBS, P.G. andLEITE, S.G.F.Departamento de Engenharia Bioquımica, Universidade Federal do Rio de Janeiro, Rio de Janeiro,RJ 21945-970, Brazil; and Departamento de Tecnologia Quımica e Departamento de Tecnologia deAlimentos, Universidade Federal Rural do Rio de Janeiro, RJ 21949-970, Brazil

(Received 14 December 1998; accepted 2 August 1999)

Abstract. The influence of temperature on growth and biomass composition of two species ofSpirulina, S. maxima and S. platensis used for food was studied. A 4L fermenter with temperature andagitation control was used to cultivate both species. Under continuous light, maximum cell production of2.4 g l–1 was verified for both cultures studied at temperatures above 25 °C: S. maxima (30 °C and 35 °C)and S. platensis (25 °C and 30 °C). An accentuated lag phase was observed for all culturesat lower temperatures (15–20 °C), and a maximum biomass production of 1.5 g l–1 was achieved. Itwas also observed that an increase of temperature caused a marked decrease in protein content, whilecarbohydrate synthesis was stimulated. The concentration of g-linolenic acid varied from 11–16% forS. maxima and from 12–14% for S. platensis, at the optimum growth temperatures. Greater culturevolumes were also studied in order to compare the performance of glass and plastic containers. Atoptimum growth temperature, S. maxima produced the same cell growth and similar final biomass com-position.

Key words: cyanobacteria, microalgae, Spirulina, Spirulina composition, temperature

Introduction

Interest in the production of algal biomass has become intense during the last50 y due to worldwide food scarcity. The cyanobacteria and microalgae such asChlorella, Spirulina and Dunaliella possess a great potential not only for theproduction of traditional food algae, but also for the extraction of valuable chemicalssuch as b-carotene and phycocyanin. Cyanobacteria, especially Spirulina, havebeen used for human feed in countries of Asia and Africa, due to their highprotein content. Spirulina cultivation is widespread in aquaculture applications due

* To whom all correspondence should be addressed at: Fax: (5221) 590-4991; e-mail:maoliveira@ufrj.br

Aquaculture International 7: 261–275, 1999.© 1999 Kluwer Academic Publishers. Printed in the Netherlands.

particularly to the use of their pigments as feed for tropical fish (Vonshak andRichmond, 1988). Microalgae are used in aquaculture as a liquid to feed young fishesand in dehydrated form to enrich foods of ornamental fish, crustaceans, shell fish andbivalves. A significant number of other algal species are used in wastewatertreatment and agriculture (Borowitzka and Borowitzka, 1988).

In their natural habitat, cyanobacteria are susceptible to sudden physical andchemical fluctuations of environmental conditions such as light, salinity, temperatureand nutrient limitation (Tomaselli et al., 1993). Temperature is one ofthe major factors controlling the multiplication of Spirulina species. Theoptimum temperature for Spirulina growth lies in the range of 30 to 35 °C,temperatures frequently encountered in North and Northeast regions of Brazil.These tropical lands are affected by the Atlantic coast and the Equator region,resulting in a humid climate with favourable conditions of temperature andlight exposure time, for algal cultivation throughout the year. These climaticconditions particularly favour the commonly practised outdoor industrial algalproduction. In many non-tropical production sites, diurnal fluctuations inpond temperatures, may be as much as 20 °C. Supplementary heating is required inwinter in temperate climates to maintain temperatures above 30 °C, thus greatlyincreasing operational costs (Bombart et al., 1993). In winter, Spirulina does notgrow significantly in open tanks (except in the tropics), resulting in lower yields(Richmond, 1992). In order to enhance culture conditions and lower those costs,algae manufacturers frequently cover the ponds with transparent polyethylene tokeep the medium warmer and free from contamination (Vonshak et al., 1992).However, despite all these precautions, cultivation of algal monoculture outdoors ishampered by contamination with other algae and with zooplankton at low tem-peratures (Vonshak et al., 1983). Low temperatures favour Chlorella cultures, whichbecome the dominant species in the culture, causing a decrease in Spirulina growth.Therefore, regions where winter temperatures will be below 15 °C are not suitable togrow Spirulina (Richmond et al., 1990).

Tomaselli et al. (1988) studied the influence of temperature on Spirulina platensisM2 cultivated continuously and observed a significant decrease in protein content(22%) associated with a remarkable increase in lipids (43%) and carbohydratecontents (30%) at 40 °C. It was also noticed at this temperature that the fatty acidcomposition changed towards a higher degree of saturation. In the same way theeffect of fermentation temperature (24, 30 and 35 °C) on the total lipid and fatty acidcomposition in supplemented cultures with linoleic acid, was studied by Quoc andDubacq (1997) who also verified that the lipid composition was highly affected bytemperature.

The objective of this study was to evaluate the influence of temperature on thegrowth of two species of Spirulina, S. maxima and S. platensis, as well as toestablish its effect on the final biomass chemical composition obtained.

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Materials and methods

Microorganisms and culture conditions. Two species of Spirulina designated Spir-ulina maxima and Spirulina platensis strains were used in this study. The cultureswere obtained from the Culture Collection of the Centro di Studio dei MicrorganismiAutotrofi of the National Research Council of Italy. Both S. maxima and S. platensiswere grown in batch culture of mineral medium, described by Paoletti (1985) (citedin Ferraz and Aquarone, 1985) with the following composition (g l–1): 2.5 KNO3,1.9 K2SO4, 0.25 MgSO4.7H2O, 0.05 CaCl2.2H2O, 0.5 K2HPO4, 15.15 NaHCO3,8.9 Na2CO3, 0.92 NaCl and micronutrient elements (to l l of nutrient solutionwere added 1 ml of: 2.86 H3BO3, 1.81 MnCl4.H2O, 0.22 ZnSO4.7H2O, 0.39Na2MoO4.2H2O, 0.079 CuSO4.5H2O, 0.049 Co(NO3).6H2O and 1 ml of Fe-EDTAsolution: 29.8 EDTA, 24.9 FeSO4.7H2O). The cultures were kept under continuouslight of 15 mmol m–2 s–1 and were transferred every month to a new medium.

Inoculum preparation. Inoculum was prepared in Erlenmeyer flasks containing100 ml of the mineral medium agitated at 200 rpm in an incubator shaker (NewBrunswick scientific, series 25D). The temperature was kept constant 35 (l) °C andillumination was provided by fluorescent lamps at an intensity of 15 mmol m–2 s–1,for 4 days.

Experiments. The experiments were carried out in a 4 l Virtis fermenter and alsoin 20 l containers made of glass and plastic with temperature control and agitation.Illumination was provided by fluorescent lamps at intensity of 180 mmol m–2 s–1. Allexperiments were initiated with 0.05 g l–1 of inoculum. Triplicate cultures wereharvested after 15 days incubation, by filtration on a 325 m mesh sieve and washedwith distilled water to remove carbonates. The water excess was eliminated using avacuum system. The biomass was transferred to a closed container, identified, frozen(225 °C) and lyophylized (LaBconco freeze dryer 18) for chemical analysis.

Experiment 1. Initially the experiments were carried out with two species ofSpirulina (Spirulina maxima and Spirulina platensis), using temperatures in therange of 15 to 45 °C, at 5 °C intervals in a 4 l fermenter.

Experiment 2. The process was carried out with S. platensis at l °C intervals,ranging from 15 to 20 °C in a 4 l fermenter.

Experiment 3. The experiment was carried out with S. maxima at a temperature of30 °C in glass and plastic containers, of approximately 20 l.

Quantitative determinations

Cellular quantification. The cell growth was measured daily by following theabsorbance variation at 600 nm with a spectrophotometer (model 20 SpetronicBausch and Lomb). The dry weight (DW) was evaluated using the relation betweendry weight and absorbance.

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pH determination. pH of the growth medium was measured daily using apotentiometer (Analion mark, model PM 600).

Total carbohydrate quantification. Total carbohydrate was quantified by themethod of Dubois et al. (1956), using glucose as standard.

Biological contaminants detection. Microscopic (Wild-M11-59128) observationswere performed in order to detect contamination with other microalgae.

Protein determination. Protein content was determined by the method of Kjeldhalaccording to Hungria and Araujo (1994).

Fatty acid profile and total lipid determination. The final biomass lipids werecontinuously extracted with petroleum ether in a Soxhlet extractor, according to thetechnique adapted by Institute Adolph Lutz (1985). After extraction, the lipids weresubmitted to saponification and methylated following the technique developed byHartman and Lago (1983). The methylated fatty acid esters were analysed in a gaschromatograph, equipped with a flame ionization detector (FID) using CP Sil 88capillary column. The operating conditions were: purge, 2 ml min–1; H2, 30 ml min–1

and synthetic air, 300 ml min–1; column temperature, 210 °C.The results of the physico-chemical analysis were calculated as mean and standard

deviations and submitted to a statistical treatment by ORIGIN SOFTWARE.

Results and discussion

Effect of temperature in the growth of S. maxima and S. platensis

Maximum cell productions 2.4 g l–1, were observed at temperatures of 30 and 35 °Cfor S. maxima and 25 and 30 °C for S. platensis (Figures l and 2). Comparing bothspecies, a wide range of temperature tolerance from 20 to 40 °C was observed. Thepreference for high temperature was evident in both species since growth was notinhibited at 40 °C and remarkably low at 20 °C. Moreover, it was observed that at20 °C, the specific growth rate of S. platensis was greater than that obtained for S.maxima. Therefore we decided to verify the growth of S. platensis in a lowtemperature range, varying from 15 to 20 °C. It was observed that microalgae growthincreased with temperature and a great reduction of metabolic activity occurred fortemperatures below 17 °C (Figure 3). Also, at temperatures of 15 and 45 °C, theabsence of growth was associated with the disappearance of green pigmentation.According to Richmond (1988) from observations of different strains of Spirulina theoptimal growth temperature was between 35 and 37 °C with 40 °C being definitelyinjurious. However, it was reported in the work of Tomaselli et al. (1988) that astrain of S. platensis could grow at temperatures above 40 °C. In a previous work,Goldman (1979) also verified that high temperature in large-scale mass culturescould lead to high yields of algae. In addition, Ogawa et al. (1971) reported that forS. platensis the optimum temperature for growth was between 35 and 37 °C.

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Figure 1. Effect of temperature on growth of Spirulina maxima in 4L glass fermenter.

Figure 2. Effect of temperature on growth of Spirulina platensis in 4L glass fermenter.

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First, the experiments were performed in glass containers of approximately 20 lusing both S. maxima and S. platensis, which demonstrated the same cell growth. Forthat reason, we decided to work only with S. maxima and have carried outexperiments in two different containers, of glass (borosilicate) and plastic (poly-carbonate), at the optimum temperature (30 °C). These tests showed the samebehaviour in terms of cell growth and final biomass composition in both containers.In this work we used large containers made of plastic, a cheaper alternative that hasnever been reported on previously. However, it proved to be an excellent alternativeas it is low cost, easy to handle and maintain, low weight and non-breakable, thusdecreasing overall operational costs (Figures 4 and 5). In microalgae cultivation foraquaculture in hatcheries, where oysters and bivalve seeds are produced, 20 l glasscontainers are most often utilized (Absher, 1998).

The existence of a great quantity of mineral salts, especially sodium bicarbonate,in the culture medium and also to a certain increase in pH, during the process, bothfavour the selective growth of Spirulina. In this work no contamination with anothermicroalgae at all range of temperatures tested was found (Figures 6 and 7). Inoutdoor cultures, Richmond (1988) reported that a pH of 11.0 is limiting to growthof Spirulina. It was further added that Spirulina can readily tolerate progressivechanges in pH; however, the culture may rapidly deteriorate when pH is changed

Figure 3. Cell growth of Spirulina platensis at low temperature in 4L glass fermenter.

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Figure 4. Cell growth of Spirulina maxima and Spirulina platensis at optimum temperature (30 °C) inglass containers.

Figure 5. Cell growth of Spirulina maxima at optimum temperature (30 °C) in glass and plasticcontainers.

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Figure 6. pH profile of Spirulina maxima at different temperatures in 4L glass fermenter.

Figure 7. pH profile of Spirulina platensis at different temperatures in 4L glass fermenter.

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abruptly, as may happen in a growth medium which is not well buffered. In thereview on Spirulina by Ciferri (1983), he mentioned that in laboratory cultures,Spirulina showed a wide range of optimum pH (8 to 11), but growth was evident alsoat pH values close to 7 and as high as 11.3.

In this work, at temperatures of 30 and 35 °C, it was verified that specific growthrate (m) values for both cultures were similar. However, S. platensis growth was lessinfluenced by temperature showing little variation on the specific growth rates(0.46–0.58 day–1) compared to S. maxima (0.26–0.45 day–1) Yet, at 40 °C, S.platensis was able to maintain a high growth rate as also observed by Tomaselli et al.(1988) (Figure 8).

Tomaselli et al. (1993), working with strains of S. platensis and S. maxima,verified that the optimum growth temperature was 35 °C. They also observed thatfive strains of S. platensis (6Mx, Sosa4, K4, Kd and M2) were able to grow attemperatures up to 42 °C, but higher temperatures were lethal. Experiments carriedout with S. platensis M2, grown at turbidostatic conditions under light limitation,showed that, following a temperature increase, the photosynthetic cell pigmentsdecreased to approximately 50% after a rise from 35 to 42 °C.

In our work it was observed that productivity increased with temperature until theoptimum growth temperature for both species was reached, and that at 40 °C, a 50%

Figure 8. Specific growth rate of Spirulina maxima and Spirulina platensis at different temperatures in4L glass fermenter.

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reduction of the value of the productivity of S. maxima occurred, and an even lowerpercentage for S. platensis was recorded (Figure 9).

Effect of temperature at the final composition of biomass of S. maxima and S.platensis

In the experiments carried out at different temperatures increasing from 20 to 40 °Ca decrease in protein content and a concurrent accumulation of carbohydrates wasobserved (Table 1). A good agreement between the final biomass compositionobtained both in glass or plastic containers was observed, as mentioned previously(Table 2). Tomaselli et al. (1993) reported similar changes in the cell macro-molecular composition as a function of temperature.

Tornabene et al. (1985), examining the lipidic and lipopolysaccharide constituentsof S. platensis, found a higher content of lipids, 16.6% in dry weight, whencompared to 11% of lipids determined by Hudson and Karis (1974) for Spirulinamaxima and 5% of lipids recorded by Switzer (1980 cited in Richmond, 1988).

Comparing the optimum growth temperature (30 °C) of both species, we observedthat the protein content in S. maxima was somewhat greater than in S. platensis,

Figure 9. Influence of temperature in the productivity of S. maxima and S. platensis.

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68.67 and 64.35% respectively (Table 1). These high protein contents indicate thatthe Spirulina species tested should be commercially available as food supplies inaquaculture. According to Schubert (1988), values of 65 to 69% protein content havealready been reported for S. maxima and 40 to 60% for S. platensis.

Effect of temperature on the profile of fatty acids of S. maxima and S. platensis

Experiment 1 temperatures in which high production of the g-linolenic acid (C18:3)was detected had been 35 and 40 °C for S. maxima and 30 °C for S. platensis (Table3). A high content of g-linolenic acid at 20 °C was also observed, probably becauseat this temperature the culture growth is still in the exponential phase as verified byTanticharoen et al. (1994). The concentrations of g-linolenic acid varied from11–16% for S. maxima and from 12–14% for S. platensis, at the optimum growthtemperatures. A high content of palmitic acid (C16:0) was found at all temperaturesfor both strains studied.

Table 1. Effect of temperature on the mean (SD) composition of the final biomass ofS. maxima and S. platensis in 4L fermenter

Temperature(°C)

Proteins (% DW)

Spirulinamaxima

Spirulinaplatensis

Carbohydrates (% DW)

Spirulinamaxima

Spirulinaplatensis

Lipids (% DW)

Spirulinamaxima

Spirulinaplatensis

20 70.24 71.56 9.88 10.58 6.22 7.24(4.84) (3.07) (1.75) (1.33) (0.37) (0.83)

25 68.01 68.04 11.68 12.65 5.97 6.32(4.35) (3.82) (0.81) (1.39) (1.27) (0.93)

30 68.67 64.35 12.72 14.35 6.20 6.96(0.68) (1.24) (1.23) (1.56) (0.50) (0.86)

35 64.58 61.63 15.57 16.24 6.79 7.18(1.19) (1.04) (0.94) (1.38) (0.24) (0.52)

40 62.81 59.41 19.63 19.93 7.30 7.24(1.30) (0.95) (1.31) (0.96) (0.59) (0.64)

The final cell concentration was 2.4 g l21.

Table 2.Composition of the final biomass of S. maxima in optimumgrowth temperature in glass and plastic containers

Glass containers Plastic containers

Proteins (%DW) 74.36 (2.23) 71.22 (3.05)Carbohydrates (%DW) 14.45 (0.97) 15.17 (0.69)Lipids (%DW) 6.91 (0.64) 8.98 (0.83)

The final cell concentration was 0.7 g l21.

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Table 3. Fatty acid profile of S. maxima and S. platensis at different temperatures

Temp.(°C)

C 16:0 (%)

Spirulinamaxima

Spirulinaplatensis

C 16:1 (%)

Spirulinamaxima

Spirulinaplatensis

C 18:0 (%)

Spirulinamaxima

Spirulinaplatensis

C 18:1 (%)

Spirulinamaxima

Spirulinaplatensis

C 18:2 (%)

Spirulinamaxima

Spirulinaplatensis

C 18:3 (%)

Spirulinamaxima

Spirulinaplatensis

20 48.03 39.73 6.41 9.11 1.62 1.29 9.00 6.63 13.12 14.77 12.87 17.61(0.56) (0.25) (0.75) (0.26) (0.13) (0.11) (0.35) (0.23) (1.15) (0.05) (0.28) (0.20)

25 45.45 44.92 6.74 6.78 1.61 1.56 11.37 11.51 1.91 12.26 13.17 14.02(0.57) (0.32) (0.05) (0.54) (0.08) (0.03) (0.19) (1.04) (0.49) (1.09) (1.19) (1.13)

30 50.42 36.38 4.46 3.39 1.79 2.76 11.17 20.92 11.65 8.69 10.96 13.65(1.60) (0.14) (0.41) (0.23) (0.23) (0.15) (1.07) (1.23) (0.51) (0.60) (0.90) (1.07)

35 47.06 46.50 3.64 2.73 1.75 2.28 8.56 14.69 13.30 11.41 15.53 12.50(0.74) (0.09) (0.26) (0.08) (0.12) (0.18) (0.75) (1.24) (1.22) (0.67) (0.82) (0.22)

40 48.66 48.77 2.06 2.11 3.03 2.19 6.75 14.48 15.53 11.36 15.09 11.22(1.33) (0.16) (0.13) (0.12) (0.29) (0.15) (0.56) (0.98) (0.57) (1.04) (0.89) (0.94)

The final cell concentration was 2.4 g l21.

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Data summarized in Table 3 shows that g-linolenic acid content does notsignificantly vary over the temperature range 20–40 °C but was not verified. Thisrepresents a great potential for industrial application of these strains because it couldmaximize protein without adversely affecting g-linolenic acid content.

Tomaselli et al. (1993) observed that, as temperature increased, lipid cell contentalso increased markedly and fatty acid composition changed towards a higher degreeof saturation; in addition, g-linolenic biosynthesis was progressively hampered andlinoleic acid was accumulated.

Several species of microalgae were selected by Franke et al. (1994) who observedhigh lipid content and a well defined fatty acid composition. Among these, cyano-phytes do not contain high fat (4.4–7.4%); fatty acid profile encountered in theirstudies were in the following range: 35–47% palmitic acid, 6–13% oleic acid, 0–27%linoleic acid and 16–23% linolenic acid.

The results of fatty acid in our cultures, presented in Table 3, show goodagreement with data reported in the literature, such as those obtained by Franke et al.(1994).

Mahajan et al. (1995) cultivated S. platensis ARM-346, Spirulina L and SpirulinaX in SOT medium, described by Hirano (1990 cited in Mahajan et al., 1995) and S.subsalsa M183 in BG-11 medium at 24 (l) °C, 120 rpm for 7 days with illuminationand verified that S. platensis accumulated large amounts of g-linolenic acid (GLA).Urea as a nitrogen source was most effective, giving a yield of 13.5 mg g-linolenicg–1 dry cell mass. Increases in temperature over the range 15 to 35 °C led to increasesin g-linolenic acid content along with biomass. Optimum temperature for maximumg-linolenic acid and biomass production was 35 °C.

Conclusions

1. S. maxima showed better growth than S. platensis at temperatures ranging from 20to 40 °C.

2. The effect of temperature on the growth of Spirulina showed a significantinfluence on protein and carbohydrate compositions, but it did not affect lipid andg-linolenic acid compositions.

3. Final biomass composition presented high nutritional value for algae cultivation,especially in sunny regions like the Northeast of Brazil, and has great potential infood applications.

Acknowledgements

We are very grateful to Dr Elioni Maria. A. Nicolaiewcksky for correcting theEnglish manuscript and Dr Eliana Flavia C. Servulo for her important suggestions

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and contributions. We also thank Dr Djalva Santana for her kindness in carrying outthe analyses of fatty acids.

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