Statistical evaluation of nutritional components impacting phycocyanin production Synechocystis SP.

Alkaliphilic cyanobacterial cultures were isolated from Lonar lake (MS, India). Among the set of cultures, Synechocystis sp, was studied for phycocyanin production. A maximum yield was obtained in BG-11 medium at optimized conditions (pH 10 and 16 h light). In order to increase the phycocyanin yield media optimization based on the eight media components a Plackett-Burman design of the 12 experimental trials was used. As per the analysis CaCl2, 2H2O and Na2CO3 have been found to be the most influencing media components at 95% significance. Further the optimum concentrations of these components were estimated following a Box Wilson Central Composite Design (CCD) with four star points and five replicates at the center points for each of two factors was adopted for optimization of these two media components. The results indicated that there was an interlinked influence of CaCl2, 2H2O and Na2CO3 on 98% significance. The maximum yield of phycocyanin (12% of dry wt) could be obtained at 0.058 g/l and 0.115 g/l of CaCl2, 2H2O and Na2CO3, respectively.

Meteoritic impact around 50-60 thousand years ago formed this crater. This is a unique crater in basaltic rock (9). Craters formed by such hypervelocity impact are considered to be a unique ecological habitat (14). The lake water remains alkaline throughout the year owing to the high content of sodium carbonate. Soda lakes are characterized by the presence of large amount of sodium carbonate (or complexes of these salts) formed by evaporative concentration under particular condition of geology, geography, and climate (7). Soda lakes are the most stable and productive naturally occurring alkaline environments in the world, with pH values greater than 10 (9).
Along with Spirulina, Synechocystis sp., a unicellular cyanobacterium gives the water a dull blue-green color.
Cyanobacteria has four basic types of biliproteins i.e.; phycocyanin, allophycocyanin, phycoerythrin and phycoerythrocyanin (16). These chromophores, called bilins, are covalently attached to the cystine residues of the apoproteins. Allophycocyanin and C-phycocyanin have a bilin called as phycocyanobilin, and C-phycoerythrin has a bilin called phycoerythrobilin (15). The absorbance coefficient of phycobiliproteins is markedly higher than those of most commercial dyes (6). This is the main reason that phycobiliproteins are used as highly-fluorescent substance in In addition, phycobiliproteins are widely used in clinical and immunological research laboratories (20).
The viability of product produced from micro-organism depends on cost of production and net yield of the product.
Media composition and growth conditions influence culture growth rate and in this case phycocyanin production. In order to avoid conventional optimization, statistical models Fractional Factorial Design (FFD) and Central Composite Design (CCD) have been proved to be effective (8,22). Here we attempt to increase the production of phycocyanin by Synechocystis sp, isolated from Lonar Lake, using methods of FFD and CCD. Media components affecting phycocyanin production were evaluated by the application of two-level factorial Plackett-Burman design (4,13).

Organism and Culture Conditions
The unicellular cyanobacterium Synechocystis sp. used in this study was isolated from the water sample of Lake Lonar.
The identification was done as per Desikachary (5). The cyanobacterium was maintained on BG-11 media (11). The pH of the media was adjusted with NaOH at 10.36 (lake water pH). In 250 ml capacity Erlenmeyer flasks 100 ml culture medium was taken and the initial cell density was maintained at 10 6 cells/ml. The flasks were exposed to a light intensity of 75 µmol photons.m -2 .s -1 with a light/dark cycle of 16/8 h at 24 ± 2°C. The irradiance was measured under the white fluorescent tube light in the culture racks using a luxmeter.

Extraction of Phycocyanin
After incubation of 12 days the cells were harvested by

Effect of pH and Light Exposure on Phycocyanin Production
In order to check the effect of pH and light exposure on phycocyanin production two sets of Erlenmeyer flasks containing 100 ml BG-11 medium at varying pH (5 to 12) and light exposure (8L:16D, 12L:12D, 16L:8D and 24L:0D h) were inoculated with Synechocystis sp. The pH of the medium was adjusted using 0.1 M sodium hydroxide and incubated at 24 ± 2°C for 12 days. After incubation phycocyanin content was measured.

Statistical Analysis
A 12-run Plackett-Burman design (13) was used to screen Phycocyanin production in Synechocystis sp.
All statistical analyses were done using statistical software MINITAB 13.31, USA, Inc.

RESULTS AND DISCUSSION
The alkaliphiles are unique microorganisms, with great potential for microbiology and biotechnological exploitation.
In present study cyanobacterial culture, Synechocystis sp. was isolated from the soda lake of Lonar, which is a unique habitat in India, rich source of naturally occurring alkaliphilic microbes.    Table 2.     Table 3. In CCD the centre point could be taken as a level that had shown maximum response in the steepest ascent experiment (21). It can be seen from Table 3 that the maximum phycocyanin production was obtained at 0.052 g/l of CaCl 2. 2H 2 O and 0.10 g/l of Na 2 CO 3 . On this basis a Box Wilson CCD (22) with four star points and five replicates at the center points for each of two factors was adopted for optimization of these two media components. Table 4 shows the observed and predicted values of phycocyanin yield obtained using CCD Equation (3).
The real values of significant variables X 4 and X 8 used in the Box Wilson model are shown in Table 5. The significant levels of each variable were determined by t-test ( Table 6). The factors, X 4 . X 4 and X 4 were found to be significant with a probability of 99% and 96.3%, respectively. The interaction between X 4 and X 8 had an interlinked influence on phycocyanin production at 98% significance.
The equation for phycocyanin yield for the second order model could be written as: = 23.6 + 1.574 X 4 + 0.564 X 8 -5.483 X 4 . X 4 + 2.528 X 4 . X 8 -    Fig. 3. It can be seen that maximum yield of phycocyanin (23.8 mg/l, i.e. 12% of cell dry weight) could be obtained at 0.058 g/l and 0.115 g/l of X 4 , CaCl 2. 2H 2 O and X 8 , Na 2 CO 3 , respectively.
In order to confirm the second order model, Synechocystis sp. was grown using optimized media constituents and yielded 22.6 mg/l of phycocyanin. The yield of phycocyanin obtained with Synechocystis sp. isolated from Lonar lake was comparable with earlier report on Synechocystis sp. PCC 6701 (25 mg/l) (8). Nitrate and phosphate were identified as the most significant factors.
The production of phycocyanin not only depends upon the media components but also some operational factors such as light and pH (8). The pH of the medium also influences the Phycocyanin production in Synechocystis sp. production of phycobiliproteins. Gloeotrichia natans, a nitrogen fixing cyanobacterium has been studied as a possible source of phycobiliproteins (3). Under optimal growth conditions (38°C, pH 8.0, with no carbon enrichment), the specific growth rate of the rice-field isolate of Gloeotrichia natans was 0.076 h -1 . The pH of the medium (between 6.5 and 9.0) did not influence the growth rate, but it did affect phycobiliprotein content, as reflected by a change of the color of the cultures. At pH 7.0, the culture was green-brown with phycobiliproteins constituting up to 10% of the total protein, while at pH 9.0 the culture was brownish-black and the content of these pigments was as high as 28% of the total protein. In the current study the optimum pH level for the production of phycocyanin was found to be pH 10 ( Fig. 1).
Phycocyanin production from cyanobacterial cultures has been reported to be dependent on various factors viz. culture conditions, particularly nitrogen and carbon sources. Supply of 3% carbon dioxide and 97% nitrogen in gaseous form supported 20.1% of Cphycocyanin yield in a laboratory culture of Nostoc sp. (19). A screening process indicated that the content of C-phycocyanin was 17% of dry weight in some strains of Anabaena and Nostoc and 10% of in some Nostoc sp. (12). A commercial process of open-tank mass cultivation of a marine cyanobacterium Phormidium valderianum BDU 30501 was also developed for producing phycocyanin (17). The average pigment yield was 20% on dry weight basis. Optimization of mass cultivation media for the production of biomass and natural colourants from marine cyanobacteria by a mathematical design of experiments were also performed (18). Kathiresan et al. (10) reported effect of major media constituents of Porphyridium spp. studied using response surface methodology (RSM) on biomass yield, total phycobiliprotein and the production of phycoerythrin. The independent variables such as concentration of NaCl, MgSO 4 , NaNO 3 and K 2 HPO 4 influenced the total phycobiliproteins and phycoerythrin production.
The production of phycobiliproteins by cyanobacteria has been observed as a pH dependent phenomenon (8). The production of phycobiliproteins increases with increase in alkaline conditions. The soda lake habitats often have a combination of Ca 2+ and carbonate levels (7). The Synechocystis sp. was isolated from Lonar Lake, a saline alkaline condition. The alkaline conditions are mainly favorable for primary producers such as cyanobacteria in the lake.