Munawer Khan Mohammed et al.

/ International Journal of Engineering Science and Technology (IJEST)

Enhancement in Production of Phenolic Compounds (AntiOxidants) in Spirulina Plantensis under Different IAA Regimes
Munawer Khan Mohammed *
Department of Microbiology, Mumtaz Degree & P.G. College, Malakpet, Hyderabad 500036, Andhra Pradesh ( India )

Mazharuddin Khan Mohd.

Prof. & Head, Dept. of Microbiology & Biotechnology Mumaz Degree & PG College, Malakpet, Hyderabad 500036, Andhra Pradesh ( India ) Abstract : The antioxidants phenolic compounds synthesis in spirulina platennsis was studied in vitro under the influence of the Indole Acetic Acid (IAA) regime. Spirulina platensis is grown in Zarrouks medium supplemented with different concentration of Indole Acetic Acid ( 1g/ml10g/ml ). The gradual increase in the total phenolic content was recorded from 1- 6 g/ml of IAA. Inhibition in the synthesis of phenolic compounds was noticed from 7g/ml-10g/ml the similar trend was also observed with chlorophyll a synthesis .Growth of Spirulina was also inhibited at higher level of IAA .Rapid maximum production of phenolic compounds noticed at 6g/ml IAA. Key words: Spirulina Platensis, Antioxidants, phenols, Indole Acetic Acid, Cholorophyll- a, Growth. INTRODUCTION: Spirulina platensis is a multicellular photosynthetic filamentous cyanobacterium (Blue green Alga) consisting of Blue green filaments of Cylindrical cells ( 1 12 m diameter ) is un branched helicoidal trichomes.It forms massive population in tropical and sub tropical water bodies which have high level of carbonate and bicarbonates with alkaline pH values of up to 11. Spirulina can colonize environments that are unsuitable for many other organisms forming population in fresh water and brakish lakes and some marine environments, mainly alkaline saline lakes (1,2). The genus spirulina has gained importance and international demand for its high value phytonutrients, pigments, which have application in health foods, feed therapeutics and diagnostics (3,4) It has been hailed as the Food of the future besides being considered as an ideal food for astronauts by NASA. It represents the most important commercial cyanobacterium (Blue-green Alga ) for the production of biomass and health foods, and animal feed (5) Recently, much attention has been focused on the micro algae, particularly Blue green micro alga ( Cyanobacteria ) as a source of novel, biologically active compounds such as phycobiline, phenols, (antioxidants) terpenoids, steroids and polysaccharide (6,7). However, the occurrence of phenolic compounds in Blue-green alga ( Cyanobacteria ) is less documented than that in higher plants. (8) Algal phenolic compounds were reported to be a potential antioxidants to combat free radicals, which are harmful to our body and food systems ( 9,10) Several epidemiological studies revealed that phenolic compounds present in diet are helpful in treating coronary heart disease (11)concern and osteoporosis stroke (12) and other degenerative diseases .Further more, phenols have been reported to exhibit pharmacological properties such as anticarcinogenic, antiviral, antimicrobial, anti-inflammatory or anti tumoral ( 13). Realizing the importance of phenolic compounds which are present in micro algae particularly in spirulina we have planned simple experiments to enhance the production of these compounds as they are the natural

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antioxidants. Hypothesized that the addition of phytohormones like Indole Acetic Acid(IAA) in nutrient media may stimulate the growth of spirulina which in turn enhance the synthesis of phenolic compounds An attempt has been made by designing simple experiment and got promising, thought provoking positive results. Materials and Methods Microorganism: Spirulina platensis used in the present study was obtained from the stock available in Centre for Food Technology and Research Institute ( CFTRI ) Mysore India. Maintenance of Spirulina platensis: The strain was grown and maintained in Zarrouks medium (14) standard synthetic medium in 500 ml Erlenmeyer flasks, maintenance & culture of the spirulina was carried out in continues Illumination at 32+/-1oC temperature the illumination provided with 6 cool white florescent tube lights 40 watts each further shaking of culture were carried out manually thrice a day. Growth: The growth of spirulina was measured every day spectrophotometrically at 540nm. Grown Spirulina culture was used for further experiments. Experimental design : Ten ( 10 )1000 ml Erlenmeyer flask were used for the experiments. In each flask 500ml of Zarrouks media was taken and under aseptic condition 10 % v/v Pure culture of spirulina platensis having an optical density of 0.34 was inoculated in each flask . After completion of inoculation Indole Acetic Acid ( IAA ) was also added in these flasks in the following manner. (Table-I)
TABLE-1: Flasks with different concentration of Indole Acetic acid(IAA)

Flask No

Zarrouks medium in ml + Spirulina 10% 500ml + Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina 500ml+ Spirulina

Concentration of IAA g/ml --1 g/ml 2 g/ml 3 g/ml 4 g/ml 5 g/ml 6 g/ml 7 g/ml 8 g/ml 9 g/ml 10 g/ml

Control Flask -1 Flask -2 Flask -3 Flask -4 Flask -5 Flask -6 Flask -7 Flask -8 Flask -9 Flask-10

Flask were incubated under artificial light provided in growth chamber and illuminated by two 4ft white fluorescent tubes which provide estimated light intensity of 15 Em2S-1(15). Samples were collected every 4th day from the flask (control and 1-10 No) to estimate the total phenols, chlorophyll-a and Growth measurement Analytical procedure 1) Total phenolic content a) Extraction of phenols : The spirulina sample were harvested and centrifuged at 2000rpm for 30min at 40 C. The pellet was resuspended in 80% ethanol and sonicated for 80sec at 600C. The residue was reextracted 5 times with 80% ethanol.(16) b) Estimation of phenols: The total phenolic content of spirulina was estimated from the extract spectrophotometerically by Folin- Ciocalteu method using tyrosine as a standard. (17) 2) Chlorophyll-a: was estimated spectrophotometerically after acetone extraction (18).

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3) Growth : Growth of spirulina was measured spectrophotometerically at 540 nm absorbance (19) Results & Discussion: Significant positive correlation between Indole Acetic Acid (IAA) concentration and synthesis of higher amounts of phenolic contents in spirulina plantensis was observed in the cells and this increased phenolic content was also related to increase in other cellular compounds such has Chlorophyll-a. (Table- 2) Modifying the culture media with Indole Acetic acid( IAA) in appropriate quantities were possibly increasing the Antioxidants potential of spirulina plantensis biomass used as a nutrient supplement. The highest quantities of phenolic compounds were obtained in the flask which was supplemented with 6 g/ml, Production of phenolic compounds increased as the concentration of IAA increased . The gradual enhancement in the production of phenolic content was noticed from4th day to 8th day. Further rapid production was noticed from 12th to 20th day . Flasks which were supplemented with 7 g/ml-10 g/ml showed decrease in the production of phenolic compounds from 12th day onwards perhaps higher concentration i.e more than 6 g/ml IAA inhibiting the growth which in turn inhibits the synthesis of phenolic compounds . The trend of synthesis of phenolic compounds were presented in the Fig-I
TABLE- 2: Effect of Indole Acetic Acid (IAA) on synthesis of total phenolic compounds

Concentration of Indole Acetic Acid 4th Day 0.8 1.2 1.5 1.7 1.8 1.9 2.4 1.2 0.9 1 1.1

Phenolic compounds in g / ml 8th Day 4 6 5.6 6.2 7.2 7.6 8 6.1 6.0 6.2 7.6 12th Day 21 24 30 32 38 41 50 35 12 11 13 16th Day 30 48 51 54 60 64 72 69 66 60 60 20th Day 60 66 69 74 78 84 96 86 80 79 78

500ml Zarrouks media + spirulina culture (control) 500ml Zarrouks media + spirulina culture + 1 g / ml(IAA) 500ml Zarrouks media + spirulina culture + 2 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 3 g / ml(IAA) 500ml Zarrouks media + spirulina culture + 4 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 5 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 6 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 7 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 8 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 9 g / ml (IAA) 500ml Zarrouks media + spirulina culture + 10 g / ml (IAA)

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Fig-1 Trend of Total Phenolic compounds production

Effect of Indole Acetic Acid on Total Phenolic Compounds

C n e tra n o P e o C m o n s in o c n tio f h n lic o p u d 120 100 80 /m g l 60 40 20 0
C o nt ro l 1 g / m l 2 g / m l 3 g / m l 4 g / m l 5 g / m l 6 g / m l 7 g / m l 8 g / m l 9 g / m l 10 g /m l

4th Day 8th Day 12th Day 16th Day 20th Day

Concentration of Indole Acetic Acid ( IAA ) g/ml

From the table-3 it is clear from that 4th day to 20th day the production of Chlorophyll a also increased in the flasks when concentration of IAA increased . Maximum chlorophyll-a content was recorded in 6 g/ml IAA containing flask, maximum production observed at 9 g/ml and decline in chlorrphyl-a was noticed in flask containing 10 g/ml IAA. From 16th day inhibition in production of chlorophyll-a was noticed in the flask containing 8 g/ml, 9 g/ml, 10 g/ml.
TABLE-3: Effect of Indole Acetic Acid (IAA) on synthesis of Chlorophyll a

Concentration of IAA g / ml

500ml Zarrouks media + spirulina culture (control ) 500ml Zarrouks media + spirulina culture + 1 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 2 g/ml (IAA) 500ml Zarrouks media +spirulina culture + 3 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 4 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 5 g/ml (IAA) 500ml Zarrouks media +spirulina culture +6 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 7 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 8 g/ml (IAA) 500ml Zarrouks media + spirulina culture + 9 g/ml (IAA) s media + spirulina culture 10 g/ml (IAA)

Concentration of chlorophyll a of Spirulina platensis (mg/10ml) 4th Day 8th Day 12th Day 16th day 20th Day 0.41095 1.30133 2.83238 3.56294 4.14748 0.9932 0.56736 0.59028 0.75717 0.91129 0.98732 0.94857 1.07481 1.02528 1.07762 1.88614 1.87819 1.87964 1.71368 1.71948 3.4438 2.42954 1.6517 1.58519 1.94301 4.3367 4.11135 5.15851 5.26571 6.23469 7.47455 6.06928 6.45487 3.30676 4.89103 4.73391 5.09506 7.55959 8.33313 9.0735 9.31214 7.55044 7.3697 5.6379 5.31274 5.67772 6.11336 8.59474 9.63276 10.65262 11.4557 8.65508 8.3267 6.9799 6.37874

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Fig-2 Trend of Chlorophyll a Synthesis

Trend of Chlorophyll - a Synthesis

C n e t aio o C o r p y - am /m o c nr t n f h lo o h ll g l 14 12 10 4th Day 8 6 4 2 0
co nt ro l

8th Day 12th Day 16th day 20th Day

Concentration of Indole Acetic Acid ( IAA ) g/ml

TABLE- IV:

Effect of Indole Acetic Acid on Growth of Spirulina platensis

Concentration of IAA g/ml 4 500ml Zarrouks media + spirulina culture (control ) 500ml Zarrouks media + spirulina culture + 1 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 2 g/ml(IAA) 500ml Zar rouks media + spirulina culture + 3 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 4 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 5 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 6 g/ml(IAA) 500ml Zarrouks media + spirulina culture +7 g/ml(IAA) 500ml Zarrouks media + spirulina culture + 8 g/ml(IAA) 500ml Zarrouks media + spirulina culture +9 g/ml(IAA) 500m l Zarrouks media + spirulina culture +10g/ml(IAA)
th

Effect of IAA on growth of Spirulina platensis at 540nm Day 8th Day 0.468 0.586 0.742 0.81 0.815 0.89 0.885 0.893 0.742 0.772 0.688 12th Day 0.59 0.901 0.951 0.788 0.92 0.998 0.747(1:1) dil 0.622(1:1) dil 0.588(1:1) dil 0.622(1:1) dil 0.547(1:1) dil 16th day 0.526(1:1)dil 0.814(1:1)dil 0.849(1:1)dil 0.996(1:1)dil 0.955(1:1) dil 0.620(1:2)dil 0.736(1:2)dil 0.687(1:2)dil 0.681(1:2)dil 0.723(1:2)dil 0.632(1:2)dil 20th Day 0.434(1:2)dil 0.670(1:2)dil 0.680(1;2)dil 0.731(1:2)dil 0.867(1:2)dil 0.846(1:2)dil 0.923(1:2)dil 0.871(1:2)dil 0.836(1:2)dil 0.833(1:2)dil 0.800(1:2)dil

0.141 0.187 0.242 0.221 0.274 0.294 0.366 0.322 0.287 0.27 0.269

The results presented in the table IV indicates that there is Concentration dependent increased in growth as recorded with phenolic content and chlorophyll-a .Further from 4th day to 20th day, maximum growth recorded in the flasks containing 6 g/ml and 7 g/ml of IAA. Then concentration dependent decline in growth was noticed in remaining flasks which are having 8,9,10 g/ml. In these flask growth decreased as concentration of IAA increased. The results indicates that addition of Indole Acetic Acid (IAA) shows the significant effect on production of phenolic compounds, chlorophyll-a and growth in spirulina . Thus its seems to be a physiological effect of IAA on spirulina as IAA stimulate the elongation of the cells, elongation occur because of increase osmotic solutes

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of the cells, reducing the wall pressure increasing the permeability of the cells to water and cell wall synthesis. Increasing the synthesis of specific DNA, new mRNA and specific enzymatic proteins which leads to stress relaxation as it is happen in the plants. These developed novel features in the spirulina enhance the uptake rate of the nutrients from the medium which leads more production of biomass. Rapid intake of nutrients leads to increase the photosynthesis in turn leading synthesis of amino acids, proteins and other cellular components. Therefore the biomass of algal cell increased in IAA containing flask, when compared with the control .These results further reveled that maximum amount of nitrogen entered in to cells of Spirulina and utilized for the synthesis of Aromatic Amino Acids (AAA) which are the precursors for the synthesis of phenolic compounds . . Inhibition in the production of phenolic compounds were noticed from 8th day onwards in the flask containing 7,8,9 and 10 g/ml of IAA. Similar trend also observed in growth and chlorophyll-a.These results indicates that decline of required nutrients in the media due to rapid uptake rate of nutrients in presence of higher quantities of IAA i.e 7,8,9,10 g/ml within a short span of time i.e.within 8 days which created scarcity of required nutrients and non-equilibrium in demand and supply in the flasks Based on the results obtained , we can conclude that Indole Acetic Acid (IAA) phytohormone can be used in appropriate quantities as a potential stimulator to enhance the production of phenolic compounds in Spirulina. Positively, in future this will become cheapest and simple method which can be adopted for the production of natural phenolic compounds (anti oxidants) in Spirulina Reference:
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