INTRODUCTION:

Microalgae are very efficient solar energy converters and they can produce a great variety of metabolites. Man has always tried to take advantage of these properties through algal mass culture. Despite the fact that many applications for algae have been described in the literature, these organisms are still of minor economic importance. ¹

In the last 50 years, algal biotechnology has shown a range of applications from the traditional extensive biomass production in human and animal nutrition (including aquaculture), soil conditioning in agriculture, technologies for bioremediation, products for the cosmetics and pharmaceutical industry, to the modern technology of high-tech, although small-scale, production of research and medical diagnostic products.

A large array of natural products of economic potential may be produced from fresh water algae. This also represent an attractive source of natural pigment such as chlorophyll, carotenoids and phycobiliprotein because of their very wide range of uses in food, feed, cosmetics and pharmaceutical area ^{2, 3}.

However, further development of this field is impeded by the high cost of the production. There is an urgent need for other strains and genera selection for obtaining high yield of biomass and special products. In this context, Oscillatoria agardhii, an allied genus of the same family, comparable to the commercial alga Spirulina platensis is being advocated for its nutritive value and pharmaceutical uses.

Biomass production in turn was depended upon several factors. The growth of any unicellular alga is a manifest of an increase in size followed by the division of the cells, which is said to be influenced by the nutritive medium, light quality and photoperiods accompanied by temperature range.^{3, 4, 1, 5}.

MATERIALS AND METHODS:

The culture of cyanobacterium O. agardhii was isolated from Mansagar Lake (Jalmahal) Jaipur, Rajasthan. Enrichment and isolations were carried out using enrichment culture media i.e. Biphasic or soil water extract media (Pringsheim’s method⁶) till unialgal forms of species were obtained. The culture was grown with photoperiod of 12 hours light/dark provided by white fluorescent lamps at a light intensity of 2,500 lux and temperature of 25 ± 2°C. Experiment to evaluate the effect of different culture media on O. agardhii was carried out in departmental laboratory.

In order to find out the best culture medium for growth and biopigment accumulation in O. agardhii, cultures were subjected to five different media of different chemical compositions, i.e. Allen and Arnon Medium ⁷, CHU-10 Midium ⁸, CFTRI Medium ⁹, Hughe’s Medium¹⁰, Zarrouk's Medium¹¹.

Three test tube sets containing 10 ml medium and 2 ml of freshly growing culture were subjected to each culture media and there growth were followed through optical density (OD) and cell count (CC). Simultaneously conical flasks containing 250 ml medium and 50 ml Oscillatoria cultures were subjected with each of these culture media for pigments (Chlorophyll-a, Carotenoids, and Phycobiliprotein) estimation.

Observations were carried out over a period of five weeks after initial readings. Growth was followed through optical density and cell count. Optical density was recorded by using colorimeter at 670 nm and cell count examination were performed using a compound microscope (Nicone SE, Japan) Cells of O. agardhii were counted in at least 10 slides from each sample using a homogeneous culture suspension.

Spectrophotometer was used for pigment estimation in different culture of O. agardhii. The chlorophyll-a content of samples were estimated by Parson and Strickland (1965) method¹², Carotenoides by Jensen (1978)¹³, and phycobiliprotein estimated by Bennett and Bogorad (1971) Method¹⁴.

Cultures were shaken gently thrice a day to avoid clumping and accelerate the growth process.

RESULTS:

Zarrouk’s Medium (pH- 10.2):

This medium supports the algal growth throughout the period of experiment. Growth rate was higher than other inorganic media but less than in CFTRI media. Optical density cell count record as well as chlorophyll-a content supported this statement. The initial optical density increase and approximately double on I week and 4.2 times on II week. Finally on V week the density was 8.2 times the initial record. Similarly cell count favours the above record and it was increased 2.21 times than the initial one (Fig. 1&2). Chlorophyll-a increase was also as per with the optical density and cell count record which showed an increase of 4.24 times the initial value on final day of observation (Fig. 2). Data pertaining to the biochemical estimation of pigments at the end of the experiment revealed 2.25% chlorophyll-a, 0.149% carotenoid, 7.38% phycobiliproteins with 4.72% phycocyanin, 2.27% allophycocyanin and 0.39% Phycoerythrin (Table-1).

Hughe’s Medium (pH-9.5):

This medium also supported the growth. However, reduced density of culture was recorded as compared to CFTRI and Zarrouk’s medium. On Iand IIweek the density increased 2.2 times and 3.5 times more than the initial record. The growth was increased linearly and finally on V week optical density increased 6.2 times than the initial value. Similarly, cell count and chlorophyll-a estimation supported the above observation. They increased linearly throughout the experimental period. On V week they were enhanced to 2.06 and 3.75 times respectively than the initial value. However, it was less than in CFTRI and Zarrouk’s solution (Fig. 1&2). After a period of Vweek culture contained 2.03% chlorophyll-a, 0.145% carotenoid, 7.41% phycobiliproteins with 4.55% phycocyanin, 2.21% allophycocyanin and 0.34% phycoerythrin (Table-1).

CFTRI Medium (pH 10.0):

Maximum growth of O. agardhii was observed in this medium. Trichomes were very active, healthy and bright blue green in colour. Fastest rate of growth was observed in this solution, when compared to other inorganic media experimented upon. In general, the growth increased linearly up to the end of the experiment. The density was more than double on IIweek. At end of the experiment cell count increased 2.29 times than the initial record. Chlorophyll-a content also supported the density records which increased gradually and finally, it was noted to be 4.5 times of the initial chlorophyll-a value (Fig. 1&2). The V week culture showed 2.51% chlorophyll-a, 0.152% carotenoid, 7.78% phycobiliproteins with 4.92% phycocyanin, 2.45% allophycocyanin and 0.41% phycoerythrin (Table-1).

CHU-10 Medium (pH-7.65):

This inorganic media was not effective in promoting the growth. Optical density increased for only II week, but decreased thereafter. Cultures turned white and did not showed any sign of recovery. Similarly, chlorophyll-a increased could be recorded up to II week but after IIIweek, it reduced drastically and particularly chlorophyll-a and other pigments could not be estimated later. By III week, pale yellow cultures had fragmented, trichomes too turned white. These cultures did not showed any sign of recovery during the entire period of observation. (Fig. 1 and 2) (Table-1)

Allen Arnon’s Medium (pH-7.3):

This medium also did not support the growth of O. agardhii but it had just support the growth for a week. After IIweek, cultures turned white and dead. Simultaneously, chlorophyll-a was also reduced and was not traceable after a period of two weeks. (Fig. 1 and 2) (Table-1)

Fig 1. Effect of Different inorganic media on the growth i.e. (A) Optical density and (B) Cell count of O. agardhii

Fig 2. Growth & bioigments of O. agardhii in different culture media

DISCUSSION:

The inorganic constituents of media have been credited to be responsible for the pattern of growth and variation in biopigment accumulation of algal forms growing either in natural or laboratory conditions. The species of cyanobacteria under investigation have been no exception to these findings.

The five inorganic media commonly used for cyanobacterial growth have been experimented upon i.e. Zarrouk’s, Hughe’s, CFTRI, Chu-10 and Allen-Arnon’s media. O. agardhii choose CFTRI for its best growth followed by Zarrouk’s, Hughe’s, Chu-10 and Allen-Arnon’s respectively. Observation was done by using the three growth parameters i.e., optical density, cell count and chlorophyll-a.

According to Droop¹⁵ more than any other factor, chemical composition of the medium may be assigned to the optimum yield of the algae. Zarrouk’s and CFTRI media were the only media to have NaHCO₃ in their composition i.e. 4.5 and 4.0 g/l respectively. HCO₃ has been used as a source of inorganic carbon^{16, 17}. Espie and Canvin ¹⁸ found that HCO₃ uptake by Synechococcus leopliensis was independent of Na⁺ concentration. Murray et al. ¹⁹ further studied the role of HCO₃accumulation with reference to photosynthesis in the above stated genus. It has a good buffering capacity for the growth medium provided by NaHCO₃. The growth was not limited neither when concentration of NaHCO₃was drastically lowered ²⁰ nor increased in S. platensis²¹.

Table 1: Effect of various inorganic media on the Biopigments of O. agardhii. Each value is the average of three replicates. Chl-a: Chlorophyll-a, Carot: Carotenoid, PC: Phycocyanin, APC: Allophycocyanin, PE: Phycoerythrin.

Week	Parameter	Zarrouk’s	Hugh's	CFTRI	CHU-10	Allen-Arnon's
Initial	Chl-a (%)	0.53	0.54	0.54	0.54	0.53
	Carot (%)	0.125	1.25	1.25	0.125	0.125
	PC (%)	3.77	3.76	3.76	3.77	3.76
	APC (%)	1.62	1.63	1.63	1.62	1.63
	PE %)	0.173	0.175	0.175	0.173	0.175
I	Chl-a (%)	0.69	0.6	0.75	0.59	0.57
	Carot (%)	0.13	0.128	1.32	0.126	0.126
	PC (%)	3.9	3.82	3.9	3.8	3.79
	APC (%)	1.75	1.72	1.84	1.69	1.66
	PE (%)	0.19	0.18	0.21	0.179	0.179
II	Chl-a (%)	1.22	0.93	1.48	0.62	0.52
	Carot (%)	0.133	0.13	0.135	0.129	0.12
	PC (%)	4.01	3.95	4.2	3.83	3.51
	APC (%)	1.88	1.86	1.95	1.72	1.04
	PE (%)	0.24	0.22	0.26	0.19	0.17
III	Chl-a (%)	1.85	1.35	2.09	0.55	0
	Carot (%)	0.139	0.136	0.141	0.112	0
	PC (%)	4.45	4.15	4.48	3.5	0
	APC (%)	1.99	1.94	2.13	1	0
	PE (%)	0.29	0.25	0.31	0.18	0
IV	Chl-a (%)	2.18	1.83	2.4	0	0
	Carot (%)	0.144	0.14	0.147	0	0
	PC (%)	4.59	4.32	4.72	0	0
	APC (%)	2.18	2.13	2.26	0	0
	PE (%)	0.34	0.32	0.37	0	0
V	Chl-a (%)	2.25	2.03	2.51	0	0
	Carot (%)	0.149	0.145	0.152	0	0
	PC (%)	4.72	4.55	4.92	0	0
	APC (%)	2.37	2.21	2.45	0	0
	PE (%)	0.39	0.34	0.41	0	0

Carbon has been known to be an essential nutrient for the production of energy and assimilation of ammonical nitrogen. However, Wissmann ²² (1991) pointed out that CO₂ showed enhanced growth more than HCO₃ as a source of carbon. But in the present species of Oscillatoria, NaHCO₃ has been found to yield standard amount of biomass (7-12 gm/sq²/day). NaHCO₃ is much cheaper than CO₂ commercially. Furthermore, the analytic grade NaHCO₃ has been replaced with locally available cheap crude NaHCO₃ commonly known as “Mitha Soda”. The luxuriant growth of these two cyanobacteria in Zarrouk’s and CFTRI medium respectively may be assigned to the NaHCO₃ present in the composition of these two media alone, as a source of carbon and a good buffering agent. The absence of this compound in the remaining solution could be one of the reasons of reduced growth of the experimental organisms.

MgSO₄ as a source of magnesium was common in most of the media. Since magnesium happened to be a constituent of chlorophyll, the algal species has an absolute requirement for this element, which contributed towards brilliant blue green coloration ^{23, 24}.

In Zarrouk’s medium phosphate in the form of K₂HPO₄, sodium as in Na₂CO_3, potassium as in K₂HPO₄ and sulphur as in K₂SO₄ were present. Whereas, in CFTRI medium the simplified crude forms of these have been added as NPK 15:15:15 agricultural fertilizer. The lack of nitrogen in the cells leads to the degradation of phycobilisomes, especially phycocyanin ^{25, 26}. Nitrogen is known to be an essential component required for protein synthesis as well as growth. Nitrate in the form of NaNO₃approximately 2.5-3.0 g/l have influenced the growth of both algae. This observation drew its support from the work of Agius and Jaccarni ²⁷ and Thomas ²⁸. Unlike nitrogen and phosphate deficiency did not result in the degradation of phycocyanin. On the contrary, phosphate was most required for the synthesis of nucleic acid, lipid and in generating high energy in the cells. Phosphate (K₂HPO₄) as a source of phosphorus has been found to be an additional requirement for the substantial growth of the two cyanobacteria.

Since, phosphorylated compounds have been suggested to be essential for metabolic activities ²⁹. Potassium inhibits the growth of several blue green algae in the absence of sodium ³⁰. However, in the present study potassium as K₂SO₄ (1g/l) in Zarrouk’s solution and NPK 15:15:15 in CFTRI medium have enhanced the growth of the algae. It deserves a special mention that K₂SO₄ is not included in the composition of the remaining media under investigation. The combination of these chemicals in the required quantity seemed to have led to the maximum yield of O. agardhii in CFTRI medium. In contrast to other solutions, these were deficient in one or the other chemicals.

The growth promoting media i.e. CFTRI solution for O. agardhii maintained normal configurations of the trichomes, while the other medium i.e. Hughe’s. Chu-10 and Allen-Arnon’s solutions showed various alterations on the morphology of the trichomes of both the species. This observation draws it support from the findings of Paraschive et al. ³¹ and Trainor et al. ³²on Scenedesmus spp.

The nature of the medium whether alkaline or acidic also depends upon the nutrients composition of the medium. This is expressed as pH of the medium, which played a significant role in the growth and maintenance of the external features of the algae. The blue green algae have generally been found to prefer pH towards alkaline side^{33, 34, 3} and present species of Oscillatoria have proved no exception to this fact. Both the genera preferred pH-10 (CFTRI) or above 10 (Zarrouk’s with pH-10.2). With lower range of pH i.e. Allen Arnon’s (7.31) Chu-10 (7.65) and Hughe’s (9.5) could not accelerate the growth of the either algae under investigation. Optimum pH for these species may be suggested as 10-10.5, Rafiqul et al. ³⁵, Smith and Wiedermann³⁶ and Sharma et al ⁵ have also established the optimum range of pH desired by varied algal species. Backer³⁷ observed that the pH determines the solubility of carbon dioxide and minerals in the medium and directly and indirectly influences the metabolism of algae.

Similar to the present findings, earlier Sharma and Kumar³⁸ have also noted different media which have offered different growth responses. CFTRI solution for O. agardhii not only rendered optimum growth but also improved the status of the cultures to brilliant blue-green. The findings of Chernyadev et al.³⁹ approved the present data that assimilation of carbon and activity of certain enzymes of photosynthesis depend on pH of the medium employed for the cultures of genus Spirulina platensis.

The culturing of the algae has been a costly affair. To minimize the cost input in the production of O. agardhii, analytical grade NaHCO₃ was replaced by commercial NaHCO₃ (mitha soda). Substitution of analytic NaHCO₃ by 4.5 g/l of cheaply available commercial NaHCO₃ in the CFTRI medium, yielded as much growth as found in analytic grade NaHCO₃ (4.5gm/l) added medium. When analytic grade NaHCO₃ (4.5gm/l) was replaced by 6.0 gm/l commercial NaHCO₃ (mitha soda) significant results were obtained because not only the growth and chlorophyll-a contents were increased but also pigments remained at their optimum level. The reason seemed to be good buffering and HCO^-₃ ions provided by mitha soda. As far as cost price of medium was concerned it was ten time less with mitha soda, as mitha soda is Rs 16/Kg against Rs 160/Kg analytic NaHCO_3.Gajraj ⁴⁰, Bhatia ⁴¹, Changothia ⁴² also reported similar results.

CONCLUSION:

More than any other factor, chemical composition of the medium has influenced the growth of cyanobacteria. The presence of sodium, nitrate, chloride, phosphate, sulphate, magnesium and carbonate has been found to be responsible for the rapid growth and high pigment accumulation in the algae ^{45, 46, 5}, on the other hand, absence of any of these ions may hamper the growth. These were very much present in CFTRI and Zarrouk’s medium.

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Received on 03.12.2012 Accepted on 02.02.2013

Research J. Science and Tech 5(2): April- June, 2013 page 239-244