THE EFFECT OF MAGNESIUM CONCENTRATION ON PROTEIN AND CHOROPHYLL a CONTENT OF Nostoc spp

Nostoc merupakan jenis blue green alga yang bisa dijadikan sumber protein potensial bagi manusia. Penelitian ini bertujuan untuk mengevaluasi kandungan protein dan klorofil a dari Nostoc yang dikultur di dua media, Modifikasi Bristol dan BG 11 media, dengan perbedaan konsentrasi MgSO4.7H2O. Di antara kedua media kultur, Nostoc yang dikultur pada modifikasi Bristol media memiliki protein dan klorofil a lebih tinggi dari Nostoc yang dikultur dengan BG 11 media. Berat kering Nostoc dengan Bristol (0.02 0.34 gram) lebih tinggi dari BG 11 (0.02 – 0.14 gram). Kandungan protein Nostoc pada Modifikasi Bristol media lebih tinggi (283.58 – 79149.77 μg/gram berat kering) sedangkan yang dikultur dengan BG 11 media (19.31 – 2536.95 μg/gram berat kering). Untuk kandungan klorofil a BG 11 media broth (0.003 – 1.67 mg/gram berat kering) lebih tinggi dari pada Modifikasi Bristol media (0.04 – 1.05 mg/gram berat kering). Perbedaan konsentrasi MgSO4.7H2O. pada kedua media kultur berpengaruh pada kandungan protein dan berat kering dari Nostoc, akan tetapi tidak berpengaruh pada kandungan klorofil a. Penambahan 0,075 gram/liter konsentrasi MgSO4.7H2O selama tiga minggu kultur merupakan kondisi optimal untuk mendapatkan kadar protein dan klorofil a yang optimal untuk modifikasi Bristol media sedangkan pada BG 11 media kandungan protein dan klorofil a yang dihasilkan optimal pada 0,000 gram/liter MgSO4.7H2O. selama satu minggu kultur.


INTRODUCTION
Prokaryotic blue green algae (cyanobacteria) are amongst the most primitive life forms on the earth. These prokaryotes share structural features with plant, such as having the ability to perform photosynthesis. Moreover, they share structural features with primitive bacteria in that they lack a cell wall. One genus of blue green algae, Nostoc, has been used for food for thousand of years. Indigenous populations in communities as diverse as the people of Japan and Hawaii, and those that occupy terrain where there are large fresh water lakes, have long recognized the value and health benefit of blue green algae in their diets.
Blue green algae is both useful and a nuisance to humans. Some spesies are regionally popular edible delicacies (Emralino & Rudolfo, 1987). Nostoc can be a nuisance on sport turf (Baldwin & Whitton, 1992), can cause unpleasant odours in drinking water and can foul buildings (Wnorowski, 1992). Human pathogens (fungal and bacterial) are inhibited by phenolic extract from Nostoc (De Cano et al., 1990): it is possible that Nostoc may be used by biotechnologist for unique medical compounds (De Mule et al., 1999). The aim of this study was to determine the effect of different concentration of MgSO 4 .7H 2 O from two kinds of culture media for protein content, and chlorophyll a content of Nostoc.

METHODOLOGY
Isolation and Purification Colonies of Nostoc: Nostoc spp. isolated from the organic paddy field soil in Udonthani province were incubated at room temperature, light intensity of 1160 lux, and 60% for moisture during one month with blue green algae nitrogen-fixing agar (Atlas, 1993).
Cultivation of Nostoc: Nostoc was cultivated in BG-11 medium and modified Bristol's solution. The micro colonies obtained from the agar plates containing specific medium culture for nitrogen-fixing blue green specific medium culture for blue green algae nitrogen-fixing agar were transferred to both of media. Transfer isolated and purified micro colony of Nostoc sp from specific medium culture for nitrogen-fixing blue green into the BG 11 and modified Bristol's solution with varied concentration of MgSO 4 .7H 2 O from 0.000 to 0.100 gram/litre (0.000, 0.025, 0.050, 0.075, and 0.100 gram/litre). Nostoc harvested every week for analyses.
Determination of Protein Content: The protein was quantified according to the colorimetric method of Bradford (1976). Nostoc crushed with mortar and pestle in NaCl 0.85%, pipeted into a test tube tube and centrifuged at 3000 rpm for 30 min. A 80 μL of supernatant of the crude protein extract was taken and put into a labeled plastic centrifuge tube before diluted in 720 μL NaCl. Drop wise add 200 μL of the Bio-Rad dye solution. The absorbance of the solution was measured at 595 nm. Plot the absorbance versus concentration of protein for each amount of BSA result were calculated and expressed as microgram of Bovine Serum Albumin (BSA) per gram dry weight of Nostoc.
Determination of Chlorophyll a Content: the chlorophyll a of Nostoc was fractionated to 80 % acetone extractable by extraction procedure. The chlorophyll a content of the fraction was evaluated using spectrophotometer. Calculations of Chlorophyll a, and Carotenoid from absorbance to mg Chl g -1 dry weight of Nostoc following Arnon's formula: Chl a (mg g -1 ) = [(12.7 x A 663 ) -(2.6 x A 645 ) x ml acetone / mg dry weight of Nostoc Chl b (mg g -1 ) = [(22.9 x A 645 ) -(4.68 x A 663 ) x ml acetone / mg dry weight of Nostoc Total Chl = Chl a + Chl b C x-c = 1000 A 470 -1. This result was expected since Nostoc cultured with different media with different concentration of MgSO 4 .7H 2 O will produce different yield. Both culture media showed increasing wet weight of Nostoc yield by the additional concentration of MgSO 4 .7H 2 O and the differences was statistically significant. Peaks of wet weight yield of MgSO 4 .7H 2 O treatments were at first week for BG 11 and at third week for Modified Bristol's solution, probably indicated reduction of magnesium content in culture media due to high magnesium requirements during growth period of Nostoc. Finkle and Appleman (1953) reported magnesium deficiency has been observed after a period of growth in different culture solutions of Chlorella vulgaris. The modified Bristol's solution can support longer growth period of Nostoc growth because it contains K 2 HPO 4 , in which has similar function with MgSO 4 .7H 2 O, that more capable to increase cell growth of Nostoc (Ogawa & Carr, 1969 Figure 2 was consistent with the percentage of dry weight content shown in Figure 1, where peaks of protein content of Nostoc cultured in BG 11 and modified Bristol's solution were 1 st week for and 3 rd , respectively. The difference of MgSO 4 .7H 2 O supplementation to the protein content of Nostoc was statistically significant, particularly in modified Bristol's solution. According to percentage of wet weight data, Nostoc cultured in modified Bristol's solution has more number of heterocyst represented by wet weight content. The divalent cation of magnesium (Mg 2+ ) has been found to induce of heterocysts formation and more hydrogen formation in Nostoc. The increase in frequency of heterocyst occurred only in the active growth phase. Induction of heterocyst could be possibly due to suppression of ammonium level, leading to enchached protein content, in case of week 1 Week 2 Week 3 Week 4 organic substances. Some energy metabolism may be linked with inorganic substances like Ca 2+ and Mg + responsible for induction of heterocyst in Nostoc and H 2 metabolism (Dawar et al., 1999).
Chlorophyll a Content: different treatment MgSO 4 .7H 2 O in the culture media did not affected to the Chlorophyll a content of Nostoc. The variation of chlorophyll a content of Nostoc varied from 0.003 to 1.67 mg/gram dry weight of Nostoc for 2 media with different concentration of MgSO 4 .7H 2 O. Nostoc cultured with BG 11 medium supplemented with high concentration of MgSO 4 .7H 2 O 0.100 gram/litre at 4 th week had much lower chlorophyll a content than concentration of MgSO 4 .7H 2 O 0.000 gram/litre at 1 st week. For the Nostoc cultured with modified Bristol's solution, the chlorophyll a content was much lower in concentration of MgSO 4 .7H 2 O 0.100 gram/litre at 1 st week than concentration of MgSO 4 .7H 2 O 0.000 gram/litre at 3 rd week (Figure 3). The difference of chlorophyll a content of Nostoc cultured in both culture media was not statistically significant, indicated that there was no effect on the variations of MgSO 4 .7H 2 O concentration. The magnesium has important function as Nostoc nutrition during photosynthesis, particularly in chlorophyll a formation. Magnesium is a central metal ion, which is bonded to a larger organic molecule called a porphyrin ring (Bohn et al., 2004) and responsible for electron transfer during photosynthesis. In this study, however, magnesium was not directly affected to the chlorophyll a content. It was probably affected by different pH in the culture media. Although we did not observed pH change during Nostoc culture and compared them among culture media, different pH during media preparation (7.1 and 6.8 for BG 11 and modified Bristol's solution, respectively) may explain inconsistent pattern of chlorophyll a data. Magnesium (Mg 2+ ) is a positively charged ion. When the pH is lowered (increase in hydrogen ion concentration or H + ), the magnesium is displaced by hydrogen ion and chlorophyll is reduced week 1 Week 2 Week 3 Week 4 because it will be converted into pheophytin which is olive green (Bohn et al., 2004).

CONCLUSION
The different concentration of MgSO 4 .7H 2 O in two cultures media affected to the dried weight and protein content, but did not affected to the chlorophyll a content of Nostoc. High content of dry weight, protein and chlorophyll a of Nostoc cultured in modified Bristol's solution might affected by K 2 HPO 4 content that has similar function with MgSO4.7H2O. Supplementation of MgSO4.7H2O with concentration 0.075 gram/litre during three week culture period was the optimum condition for Nostoc cultured in the modified Bristol's solution that produces optimum protein and chlorophyll a contents, while BG 11 will produce optimum protein and chlorophyll a contents of Nostoc in concentration 0.000 at first week. This study provides baseline data for culture development and nutritional value of Nostoc for food supplement use.