An assessment of biopotential of three cyanobacterial isolates from Antarctic for
carotenoid production
S P Shukla1* and A K Kashyap2
1Department of Botany, Arunachal University, Rono-Hills, Itanagar 791 111, India;
2Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221 005, India
Received 25 February 2003; revised 4 September 2003 Specific growth rates and carotenoid contents of three Antarc- tic and tropical strains of cyanobacteria viz. Anabaena sp., Phor- midium sp. and Nostoc sp. were compared in batch and mass cul- tures to assess bio-potential of Antarctic strains for cost-effective carotenoid production. Antarctic strains though exhibited slightly lower specific growth rates, but contained higher carotenoid con- tents (per unit dry wt.), than tropical strains. Modification of nor- mal composition of BG-11 culture medium, by altering nitrogen and carbon sources resulted in 25-38% increase in carotenoid content in both types of strains. Mass-culture in indoor and semi- outdoor bio-reactors resulted in 39-113% higher carotenoid con- tent in Antarctic strains, compared to their respective tropical strains. The observations suggest that Antarctic cyanobacteria may have potential as superior strains for maximizing the yield of carotenoids.
Key words: Antarctic, cyanobacteria, carotenoids, mass-culture
Carotenoids are widely used as natural colorant for food, drug and cosmetic products1,2. They serve as a second line of defense against oxidative modification of low density proteins3,4. Dietary intake of carote- noids is positively correlated with chemoprevention of cancer and other degenerative diseases5-7. The vari- ous carotenoids are also found to enhance effector Stage T helper cell activity, which is an important component of immune system8. Earlier, carotenoids have been reported in volvocalean alga Dunaliella salina9,10 and some cyanobacterial species viz., Phor- midium laminosum11, Synechococcus sp. (strain PCC 794212,13) and Nostoc commune14. However, reports pertaining to the biopotential of cyanobacteria of Ant- arctic origin are lacking. Further, a long gap exists in knowledge regarding the optimization of growth con-
ditions for enhanced carotenoid production by Antarc- tic strains under simulated conditions. In an earlier, preliminary study, we observed that Antarctic strain of Anabaena synthesized approx. 2-folds higher caro- tenoids and 3-folds higher phycocyanin, compared to the respective tropical strain, when grown in Chu-10 medium at 5°C under light intesity 45 µE15. In the present paper, the comparative study of specific growth rates and carotenoid contents of Antarctic and tropical strains of three cyanobacteria viz. Anabaena sp., Nostoc sp., and Phormidium sp. is reported. The observations were recorded in unialgal populations of above strains, grown in batch and indoor and semi- outdoor mass culture units. The growth medium (BG- 11) was also modified for further increase in the yield of carotenoids.
The Antarctic isolates of Anabaena, Nostoc and Phormidium were isolated from the algal patches col- lected in Antarctica from a glacial melt water stream (70o46'05"S, 11o43'15"E) situated near "Maitri" (Per- manent Indian station) in Schirmacher Oasis, Antarc- tica. The oasis is oriented in east-west direction with a coordinates of 70o46'04"S to 70o44'2"S; 11o49'5"(+48) to 11o26'03(+02) E. Average annual temperature of the oasis for the period 1961 to 1980 was −10oC. The samples collected from the site were brought to greenhouse of permanent station "Maitri" in sterilized polythene bags and inoculated on semi solid (agar base) BG-11 culture medium. Average temperature inside the greenhouse ranged between 12°-15°C. Fol- lowing incubation for 15 days under photo- autotrophic growth conditions, the enriched colonies appearing on the plates were transferred to 5 ml BG- 11 liquid culture medium. After suitable growth, the culture suspension was inoculated on agar slants (BG- 11) and slants were incubated in air-conditioned cabin of ship during journey.
Tropical isolate was isolated from the heterogene- ous algal assemblages growing in rice field of agricul- tural farm of Banaras Hindu University (B.H.U., Va- ranasi, India). Tropical isolate was chosen for com- parative study on the basis of its affinity with Antarc- tic isolate in terms of morphology and growth behav- iour. The Antarctic and tropical isolates were cultured by employing standard microbiological techniques16.
The normal composition of the medium was altered
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*Author for correspondence E mail: sps_s@rediffmail.com Fax: 0360-2277317
by decreasing the concentration of NaNO3 and K2HPO4 and increasing the concentrations of citric acid (2-folds). In modified medium, MgSO4⋅7H2O and Na2CO3 of normal medium were replaced with MgCl2 (0.10 g/L) and NaHCO3 (0.05 g/L) and apart from NaNO3, one more combined nitrogen source, NH4Cl (0.026 g/L) was added. The composition of normal and modified culture media is given in Table 1; pH of the modified medium was adjusted to 7.1, by adding 20 mM HEPES-NaOH buffer.
Carotenoids were extracted and determined by the method as described17. Total pigments were extracted in acetone, dried completely and dissolved in ethanol and then treated with ethanolic KOH at 65°C. An equal volume of salt-saturated water was added and the solution was partitioned against diethyl ether. Ca- rotenoids in the ether phase were recovered by drying under stream of N2 and dried carotenoids were dis- solved in a minimal volume of ethanol. Concentration of carotenoids (µg/ml) was calculated according to formula given below.
Carotenoid (µg/ml) = [O.D.461-(0.046 × O.D.664] ×4 The sample containing 25-50 ml algal suspension was filtered through Whatman GF/C filter (47 mm diam.), and dried in oven for 24 hr at 70°C, prior to filtration. The sample, while filtered, was washed with 20 ml acidified water (pH 4) to remove/wash algal cells from insoluble salts. The filter was put in a glass petri-dish in the oven under the above condi- tions and weighed after being cooled in a desiccator.
Two types of mass culture assemblies (a) Indoor
mass assembly; and (b) Semi outdoor culture assem- bly were used.
Indoor mass culture unit consisted of a glass vessel (20 L capacity) fitted with an air compressor (Fig. 1).
The flow of air was regulated with an air flow meter.
The air was passed through a water bath (consisting of 0.1% KMnO4) and air filter (sintered glass), prior to release in the growth medium. A known quantity of algal cell-mass was inoculated to obtain an initial cell density equivalent to 150 µg protein ml-1. The culture was stirred with a magnetic stirrer and light (15 wm-2) on the surface of culture vessel was provided at 12:12 hr light:dark periods. Specific growth rates (k) were recorded on alternate days, by collecting a small vol- ume (10 ml) of the culture suspension from the glass- stoppered outlet of culture vessel. The values of k were calculated by method and formula given ear- lier18.
Semi-outdoor unit consisted of two subunits (i) in- door culture tank of 30 L capacity fabricated from transluscent perspex sheets; and (ii) outdoor tubular unit connected to the indoor culture tank through an inlet and outlet forming a closed system (Fig. 2). The culture was constantly stirred with the help of a mag- netic stirrer and fresh air was passed through a device, similar to that of indoor culture assembly. The indoor tank containing fresh culture medium was inoculated with a cell mass equivalent to 100-150 µg protein ml-1, by harvesting exponential phase population batch cultures.
The culture suspension of the indoor culture tank was circulated through the polypropylene tubes (1/2"
diam.) connected to the inlet and outlets of the indoor
Fig. 1—Culture assembly for mass cultivation of cyanobacteria (indoor)
culture tank. The tubes were encircled on a hollow metallic frame lying outside the laboratory at a posi- tion, where normal sunshine was available throughout the day. All the experiments were carried out in the month of January 1998, when temperature fluctuated between 20-24oC.
A significant increase in specific growth rate (k) was recorded in the populations grown in modified BG-11 medium (Table 1); maximum increase was observed in Anabaena of Antarctic origin, which showed 16.6% higher specific growth rate in modified medium, as compared to the normal medium. In gen- eral, Antarctic and tropical strains exhibited 10-17%
increase in specific growth rates in modified medium (Table 2).
Carotenoid content in Antarctic and tropical strains increased in the range of 20-38% in modified me- dium, than the cultures grown in normal medium (Table 3). Antarctic strain of Anabaena showed an increase of 38% in carotenoid content in the modified medium, followed by tropical Nostoc sp. (27.7%).
The content was higher by 112.7, 85.1 and 36.5% in Antarctic strain of Anabaena, Nostoc and Phormi- dium, respectively (Table 3).
Carotenoid contents in Antarctic and tropical strains were directly influenced by the method of cul- tivation. The carotenoid contents of cyanobacterial strains grown in batch and mass-cultures (indoor and semi-outdoor) are given in Table 3; maximum in- crease was observed in Antarctic strain of Phormi- dium sp., grown in semi-outdoor culture unit, where an increase of 72-113% was recorded, as compared to batch-cultures. The populations grown in indoor cul- ture unit also showed an appreciable increase (39- 70%) in carotenoid content, compared to batch- cultures.
Table 1—Composition of normal and modified BG-11 medium (Allen, 1968) (gL-1)
BG-11 BG-11
(Normal) (Modified)
NaNO3 1.5 0.8
K2HPO4 0.04 0.01
MgSO4.7H2O 0.075 -
MgCl2 - 0.10
CaCl2.2H2O 0.036 0.036
Citric acid 0.006 0.012
Ferric ammonium citrate 0.006 0.002
Na2-EDTA 0.001 0.001
Na2CO3 0.02 -
NaHCO3 - 0.05
NH4Cl - 0.026
Trace elements were same for modified and normal medium Table 2—Specific growth rates of six strains of cyanobacteria in
normal and modified BG-11 growth medium [Values in parenthesis indicate generation times (hr)]
Organism Specific growth rate (k)
BG-11 (Normal) BG- 11 (modified) Anabaena sp. (Ant.) 0.36(66.6) 0.42(57.1) Anabaena sp. (Trp.) 0.39(61.5) 0.44(54.5) Nostoc sp. (Ant.) 0.41(58.5) 0.46(52.2) Nostoc. sp. (Trp.) 0.42(57.1) 0.48(50.0) Phormidium sp. (Ant.) 0.38(63.2) 0.43(55.8) Phormidium sp. (Trp.) 0.41(58.5) 0.45(53.3) Ant, Antarctic; Trp, tropical
Fig. 2—Semi-outdoor culture assembly for mass cultivation of cyanobacteria
Table 3—Carotenoid contents of cyanobacterial strains in normal and modified BG-11 culture medium and batch and mass cultures
after six days of growth
Carotenoid content (µg.mg dry wt.) Organism BG-11 BG-11 Batch Mass culture
(Nor- mal)
(Modi- fied)
Cul- ture
I SI
Anabaena sp.
(Ant.)
14.2 19.6 18.8 26.2 32.4
Anabaena sp.
(Trp.)
7.3 8.8 9.2 14.4 16.8
Nostoc sp. (Ant.) 12.6 16.1 17.6 29.9 36.4 Nostoc sp. (Trp.) 6.8 8.7 8.4 13.2 16.4 Phormidium sp.
(Ant.)
11.4 14.2 15.2 24.6 32.4
Phormidium sp.
(Trp.)
8.2 10.4 11.4 18.8 22.6
Ant, Antarctic; Trp, Tropical; I, Indoor; SO, Semioutdoor
Among Antarctic strains, maximum yield of caro- tenoids was obtained in Nostoc sp. (36.4 µg.mg-1 dry wt.), followed by Anabaena sp. (32.4 µg.mg-1 dry wt.). Among the tropical strains Phormidium sp.
showed highest content (22.6 µg.mg-1 dry wt.) in semi-outdoor unit (Table 3), which was 43.4% lower than Antarctic strain grown under similar conditions.
In semi-outdoor mass-culture, the carotenoid content of Antarctic strains was higher by 92.85, 121.9 and 43.4%, as compared to the tropical strains of Ana- baena sp., Nostoc sp. and Phormidium sp., respec- tively.
The differential response of Antarctic and tropical strains in terms of growth rate and carotenoids content indicates that physiological and biochemical proper- ties of Antarctic strains differ considerably with tropi- cal strains, under a given set of growth conditions.
Owing to extreme environment (prolong light and dark periods, higher intensity of UV-radiation, super- saturation of water with oxygen etc. of Antarctica), Antarctic strains may have developed adaptations in their metabolic set-up to survive in the inhospitable conditions. Earlier studies19-21 have shown that meta- bolic processes viz., nutrient uptake, nitrogenase, glutamine synthetase and alkaline phosphatase activi- ties differ considerably in Antarctic and tropical strains in the temperature range 5°-40°C. Thus, slower growth and higher carotenoid contents of Ant- arctic strains seem to be a manifestation of differences in their metabolic functions.
An increase in growth and carotenoid content in Antarctic strains, compared to the batch cultures may be attributed to constant aeration and stirring of cul- ture suspension in indoor and semi-outdoor mass cul- ture. Besides, in indoor mass culture assembly, the area receiving light was larger, compared to the batch cultures where penetration of light was lesser, due to conical structure of flasks. Earlier reports22,23 have shown that in mass-culture of cyanobacteria, open culture systems are more susceptible to contamination than closed systems which decrease the market value of algal biomass/or derived products produced in open systems. Thus, there is an emphasis on cost-effective closed systems for mass cultivation of cyanobacteria.
The semi-outdoor bioreactor used in present study had an advantage over indoor closed reactor, because out- door portion of reactor received natural light, there- fore, the cost of light assembly was reduced. Further- more, the temperature of culture medium could also be regulated from inside the laboratory, using a tem- perature-controlled water-bath. The circulation of the culture suspension in tubular portion further reduces the cost incurring on stirring.
Antarctic and tropical isolates exhibited best growth and carotenoid contents in the cultures grown in semi-outdoor mass culture assembly which may be due to (i) better availability of light to the assembly where cultures circulated in the tubular portion were exposed to natural light conditions; and (ii) continu- ous circulation and mixing of culture suspension in tubular outdoor portion results in better aeration in comparison to batch and indoor mass cultures.
The present study suggests that Antarctic strains of cyanobacteria have a potential for commercial pro- duction of carotenoids. Though, they exhibited a slower growth, compared to the respective tropical strains, but their carotenoid content (per mg dry wt.
basis) was appreciably higher than tropical isolates.
The carotenoid content in Antarctic strains was 5-6- folds higher than reported in one of the commercially viable strain Spirulina maxima24. However, further studies are required for better assessment of biopoten- tial of large number of other Antarctic cyanobacteria for carotenoid extraction.
Authors are thankful to Department of Ocean De- velopment (D.O.D.), New Delhi for the support dur- ing the Indian Antarctic Expeditions (6th and 15th).
SPS is thankful to CSIR, New Delhi for financial as- sistance.
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