Effect of Oxygen Concentration on the Protein Content of Spirulina Biomass

G. Torzillo, L. Giovannetti, F. Bocci, and R. Materassi Centro di Studio dei Microrganismi Autotrofi del C.N.R. c/o lnstituto di Microbiologia agraria e tecnica Piazzale delle Cascine, 27 50144 Firenze, Italy

Accepted for Publication February 14, 1984

Research in progress in this laboratory on the outdoor mass culture of Spirulina platensis and S. maxima have shown that the chemical composition of Spirulina bio- mass changes more or less markedly according to the type of reactor in which the organism is grown. For both spe- cies, the biomass grown in closed reactors made of 14- cm-diameter polyethylene tubes (total length 400 m), in which the culture suspension is circulated with a peris- taltic pump at a speed of about 3 cm/s, has a protein con- tent lower than biomass produced in open “raceway” ponds provided with paddle wheels for the circulation of the culture suspension. It was also observed that the pro- tein content of Spirulina grown in closed tubular reactors increased somewhat when the circulation speed of the culture was increased or when the total length of the tu- bular circuit was decreased. In both cases the time re- quired by the culture to cover the entire tubular circuit was lowered.

Since during the stay inside the polyethylene tubes, photosynthetically produced oxygen accumulates in con- siderable amounts, the observations reported above led us to investigate the influence of oxygen tension on the protein content of Spirulina biomass.

EXPERIMENTAL METHODS

The experiments were made in 500-mL Pyrex bottles (Sovirel) containing 300 mL of the standard culture solu- tion employed in outdoor mass culture of Spirulina. The bottles were hermetically sealed with a silicone mem- brane and a screw cap. Each bottle was inoculated with about 40 mg cells (dry wt) taken from a two-day-old cul- ture on the same medium.

The oxygen concentration in the atmosphere was ini- tially adjusted at the desired level. Inoculated bottles were incubated in a thermoluminostatic chamber (Psy- chrotherm G 26, New Brunswick Scientific) on an orbital shaker (100 rpm) under a light intensity of 7.000 lux, measured with a LICOR Quantum Radiometer-Photom- eter model LI-l85A, from cool white fluorescent lamps placed above the bottles.

Continuous turbidostatic culture experiments were

Biotechnology and Bioengineering, Vol. XXVI, Pp. 1134-1 135 (1984) 01984 John Wiley &Sons, Inc.

carried out with a rectangular culture chamber (15 X 30 X 5 cm) with glass walls. The desired O2 concentra- tion was obtained by bubbling an appropriate amount of filter sterilized air into the culture. The 0 2 concentration in the culture was monitored polarographically with a Dissolved O2 meter (model 1010, Delta Scientific Corp.).

Oxygen concentration in the gas phase of the bottles was determined with a gas chromatograph (Perkin- Elmer, model Sigma-2) equipped with Porapack Q columns (2 m length and 2 mm width). Gas carrier N2 was used. The temperatures of the injector, detector, and oven were 150, 150, and 4SoC, respectively.

Chlorophyll a , phycocyanin, and total carotenoids were assayed following the procedures described by Par- son and Strickland,’ and O’Heocha,2 and Paoletti and co -~orke r s .~ Protein content and amino acid composition were determined with a model 31\27 amino analyzer (Carlo ErbaL4

RESULTS AND DISCUSSION

While the biomass yield was not significantly affected by O2 partial pressures from 0.20 to 0.56 atm, the protein content was markedly reduced, particularly when the ini- tial O2 concentration was raised to 0.45 atm (Table I). The exposition to an atmosphere containing 70% O2 markedly reduced the yield of biomass and almost com-

Table I. Effect of oxygen tension on the growth and protein and pig- ment content of Spirulina maxima 4Mx after five days culture at 30°C in sealed bottles.

Initial O2 concentration (9’0 in the gas phase) Controla 20 45

O2 concentration after five days (TO in the gas phase) 43 56

Biomass yield (mg/100 mL culture) 63 66 60 Protein content (% dry wt) 48 33 22 Chlorophyll (mg/g dry wt) 11.8 10.9 9.4 Phycocyanin (mg/g dry wt) 60 35 18

a Culture was kept in cotton stoppered bottles.

CCC 0006-35921841091 134-02$04.00

Table 11. in continuous turbidcstatic culture.

Influence of temperature and oxygen concentration on protein and pigment content of Spirulincr nztrxirnu 4 M x grown

Dry weight 0, concentration in

the culture Temperature Phycocyanin Chlorophyll Carotcnoids (ppni) ("C) Protein (mg/g) (mg/g) (mg/g)

9 9

15 15

20 63 1 66 22 5.4 30 613 66 22 5.2 20 226 24 12 2.3 30 428 51 21 2.7

pletely inhibited protein synthesis. Chlorophyll content was scarcely affected while phycocyanin content was strongly reduced.

The amino acid pattern of the protein fraction of cells exposed to an high oxygen concentration underwent some modifications. A decrease in aspartic acid and an equivalent increase in alanine contents were observed. A considerable reduction occurred in the sulphur-contain- ing aminoacids.

Continuous culture experiments with a turbidostatic apparatus have shown that the effect of oxygen tension on the protein content of Spirulina biomass is temperature dependent (Table 11). At 20°C the inhibition of protein synthesis is much more pronounced than at 30°C. An in- crease in O2 concentration similarly decreases the content of photosynthetic pigments.

Under our experiment a1 conditions, the phycocyan in content was unaffected by temperature in the range 20- 30°C. Thus, Spirulina maxima 4Mx differs from Anacys- tis nidufuns, whose phycocyanin was 20% lower at 25°C than at 35"C.s

Photooxidative death in cyanobacteria exposed to high light intensity and high oxygen concentration in media devoid of C02 has been d e ~ c r i b e d . ~ . ~ On the other hand, in the presence of C 0 2 , high oxygen concentrations can

induce a bleaching of photosynthetic pigment^.^-^ Our experiments have shown that O2 tensions moderately higher than 0.2 atm can be without effects on the growth rate of Spirulina spp. grown in a bicarbonate-rich me- dium. However, the protein content of the biomass de- creased considerably. Since these conditions are likely to occur in large-scale cultures, the phenomenon reported here must be considered in designing systems for the mass cultivation of Spirulina.

References

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2. O'Heocha, in: Chemistry and Biochemistry of Plant Pigments.

3 . C . Paoletti, G . Florenzano, and W. Balloni,Ann. Microbiol., 21, 71

4. C . Paoletti, G . Florenzano, and R. Materassi, Sci. Tecnol. A l im. , 3 ,

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