International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 1(6), 49-56, October (2012) I. Res. J. Biological Sci. International Science Congress Association 49 Impact of different Physical and Chemical Environment for mass Production of Spirulina pletensis- An Immunity Promoter Mehta Jitendra*, Sharma Priyanka, Jakhetia Madhulika, Syedy Mohsina, Makhijani Komal and Khamora Neha Algae Research Laboratory and Department of Biotechnology, Vital Biotech Research Institute, University of Kota, Kota, Rajasthan, INDIAAvailable online at: www.isca.in Received 16th August 2012, revised 24th August 2012, accepted 28th August 2012Abstract Spirulina is one of the most explored cyanobacteria. Since ancient time it is being used as source of protein. Spirulina pletensis was cultivated in different medium like; zarrouk’s medium, CFTRI medium, OFERR medium, zarrouk’s medium + PGR medium, agitation and without agitation medium, improved VITAL BIOTECH medium. Different temperature, light intensity and pH were monitored for 20 days on daily basis. pH was found in range from 9.1 to 10.1in different medium. Gradually increase in dry weight (dw) was noticed along with the age of culture, 0.40-1.25 dw/l was achieved in different medium respectively. Spirulina inoculated in improved VITAL BIOTECH medium was survived and growth was flourished, achieving dry weight of 0.82 dw/l on 20th day of cultivation. Different amount of NaHCO and NaNO3 shown significant impact on Spirulina growth. However results of present investigation could be consider for commercial cultivation of Spirulina using different physical and chemical environment for mass production of Spirulina pletensis. Keywords: Cyanobacteria, spirulina pletensis, CFTRI media, OFERR Media. IntroductionThe potential of cyanobacteria to produce large number of chemicals and biological compound such as vitamins, carotenoid pigments, proteins, lipids and polysaccharides. Placed cyanobacteria in the list of quite interesting microorganisms for commercialization purpose of cyanobacteria is cultivated in large amount. Researchers are also doing this on global level. Spirulina is cultivated in tropical and subtropical bodies of water and filamentous form of cyanobacteria. The water bodies should have high levels of carbonate and bicarbonate and alkaline Ph values of up to 11. In Africa chad lack in maxico texcoco lack produces spirulina which is harvested as a source of food. Spirulina is singal cell protein to produce vitamins, minerals, proteins, and polyunsaturated fatty acid. Therapeutic properties, antioxidant activity, tubular photobioreactors, glass panels are used to cultivation of cyanobacteria. Cost and composition media is challenging factors for viable production of cyanobacteria. Different media used for cultivation of spirulina such as Zarrouks media, Rao’s media, CFTIR media, OFERR media Revised media (6) and Bangladesh medium. The present report is aimed to the study of impact of different physical and chemical environment for mass production of spirulina pletensis. Spirulina is type of filamentous blue green alga due to the capacity to produce bioactive components such as vitamins, minerals, polyunsaturated fatty acid, carotenes, and other pigments that have an antioxidant activity to receiving attention spirulina have a antioxidant capacities to attribute biliproteins called as phycocyanin. Proteins (60%-70%), vitamins, essential amino acid, minerals and essential fatty acid such as palmitic acid, linolenic acid and linoleic acid are produced by sprulina. Spirulina is used as nutrient source for fish larvae. It is used in fish diet as an ingredient for juveniles and adults common corp. Material and MethodsCulture development and maintenance: The strain of Spirulina pletensis was obtained from IARI, New Delhi, which is previously maintained in Zarrouk’s agar media slants in 4ºC. Spirulina pletensis was grown in Zarrouk’s medium. Firstly, we have transferred our culture in Zarrouk’s broth from Zarrouk’s agar slants. Culture were incubated in a culture room at temperature of 30±2ºC and illuminated with day-light fluorescent tubes saving 4 Klux at a surface of vessels. During the process of growth the flask was shaken 3 to 4 time per day. The experiment was run in triplicates. All manipulation involving the transfer of culture in the liquid media or on agar plates were carried out under aseptic conditions on a laminar air flow. Preparation of Zarrouk’s media as following step: i.Firstly we had taken sterilized 1000ml conical flask. ii. Then we had taken 500ml D/W in flask. iii. After that mix properly all components of Zarrouk’s media. iv. Autoclaved 500ml media for 15 to 20 minutes. Preparation of inoculums: Inoculum preparation for culture maintenance taken well-developed biomass concentration of Spirulina culture, which has inoculated before 20 to 25 days in Zarrouk’s media. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 49-56, October (2012) I. Res. J. Biological Sci. International Science Congress Association 50 Filtration: Cells were collected by filtration using filter paper 8mm pore size (Screen printing paper). Washing: Cells were washed with buffer solution (pH 7), diluted to known volume and processed for further inoculation. Shaking in cyclomixture: Diluted inoculum shaked in cyclomixture for making homogenized mixture. Dry weight measurement: For dry weight measurement homogenous suspension of known quantity of Spirulina sample were filtered through screen-printing paper and oven dried at 75ºC for 2 to 6 hours. The dried filter paper containing Spirulinabiomass were cooled and weighted. The difference between the initial and final weight were taken as the dry weight of Spirulinabiomass. The dry weights were expressed in terms of gm/litre. Results and DiscussionGrowth results expressed in terms of dry weight of S. pletensisat different physical and chemical parameters viz. temperature condition-25\rC and 35\rC (room temperature) (table-1) described maximum bulk density (0.60gm) of S. pletensis obtained at 25\rC temperature 1200 lux. Subsequentlyon different light intensity viz. 435 lux, 650 lux, 975 lux, 1100 lux, 1300 lux in flask (table-2) described maximum bulk density (0.80gm) of S. pletensis in 1300 lux. Then Change in concentration of Carbon and Nitrogen source (table-3) described maximum bulk density of S. pletensis in 1.0 gm of NaNO (0.89 gm) and 8 gm of NaHCO3 (1.25gm). After thatat different pH (7, 8, 9, 10, and 11) in flask culture showed the maximum bulk density about 0.81gm/ml when pH of culture medium was maintained at 11 with medium volume 250ml in a 500ml flask (table-4).And in further parameter of Growth results of S. pletensis at different medium viz. CFTRI, Bangladesh, RM-6, A-5, and OFERR showed maximum growth of S. pletensis in a flask with RM-6 medium volume 250ml in a 500ml flask (table-5). Finally S. pletensis was grown at different chemical and physical environment in flask culture viz. Zarrouk’s Media+PGR, Zarrouk’s Media, Agitation Zarrouk’s Media, Without Agitation Zarrouk’s’ Media. Maximum growth of S. pletensis was noticed a flask which has Agitation Zarrouk’s Media with medium (table-6). Table-1 Biomass production of Spirulina pletensis different different temperature Temperature in Microorganism (Spirulina pletensis) (Temperature 25 \rC, Duration 3 weeks) Initial pH 8 of Zarrouk’s Media Final pH of Zarrouk’s Media 25 Biomass (gm/ 250 ml)Frequency of biomass Growth (%) Color9.82 0.60 80 dark green, thick few clumps, no contamination 350.49 65 green, thick few clumps, no contamination 9.80 Table-2 Biomass production of Spirulina pletensis different light intensity Intensity of light in lux Microorganism (Spirulina pletensis) (Temperature 25 \rC, Duration 3 weeks) Initial pH 8 of Zarrouk’s Media Final pH of Zarrouk’s Media 435 Biomass (gm/ 250 ml)Frequency of biomass Growth (%) Color9.41 0.70 65 light green, thick few clumps, no contamination 650 0.67 60 green, thick few clumps, no contamination 9.38 975 0.65 57 light green, thick few clumps, no contamination 9.23 1100 0.75 70 Dark green, thick few clumps, no contamination 9.79 1300 0.80 80 Dark green, thick few clumps, no contamination, thin film of cells on flask wall 9.90 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 49-56, October (2012) I. Res. J. Biological Sci. International Science Congress Association 51 Table-3 Biomass production of Spirulina pletensis different carbon and nitrogen source Different Carbon Source NaHCO3 gm/250ml Different Nitrogen Source NaNO3 gm/250ml Microorganism (Spirulina pletensis) (Temperature 25 \rC, Duration 3 weeks)Initial pH 8 of Zarrouk’s MediaFinal pH of Zarrouk’s Media 3.0 - Biomass (gm/ 250 ml) Color Frequency of biomass Growth (%) 10.1 0.74 light green, thick few clumps, no contamination 60 8.0 - 1.25 green, thick few clumps, no contamination 85 10.0 10.0 - 1.11 Dark green, thick few clumps, no contamination, thin film of cells on flask wall 78 9.9 - 0.037 0.76 pale green, no contamination 62 9.9 - 0.5 0.80 green, thick few clumps, no contamination 66 10.0 - 1.0 0.89 Dark green, thick few clumps, no contamination 70 10.0 Table-4 Biomass production of Spirulina pletensis in different pH Initial pH of Zarrouk’s Media Microorganism (Spirulina pletensis) (Temperature 25 \rC, Duration 3 weeks) Final pH of Zarrouk’s Media 7 Biomass (gm/ 250 ml) Frequency of biomass Growth (%) Color9.23 0.73 70 light green, thick few clumps, no contamination 8 0.76 75 green, thick few clumps, no contamination 9.30 9 0.70 65 green, thick few clumps, no contamination 9.67 10 0.77 80 Dark green, thick few clumps, no contamination 9.68 11 0.81 90 Dark green, thick few clumps, no contamination, thin film of cells on flask wall 9.88 Table-5 Biomass production of Spirulina pletensis different medium Different media Microorganism (Spirulina pletensis) (Temperature 25 \rC, Duration 3 weeks) CFTRI Biomass (response)Color Frequency of biomass Growth (%) +++++ green, thick few clumps, no contamination70 Bangladesh ++ Pale green, no contamination 40 RM-6 +++++++ Dark green, thick few clumps, no contamination, thin film of cells on flask wall 80 A-5 - - - OFERR +++ light green, no contamination 60 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 49-56, October (2012) I. Res. J. Biological Sci. International Science Congress Association 52 Table-6 Biomass production of Spirulina pletensis in different chemical and physical environment Different media Microorganism (Spirulina pletensis) (Temperature 25 \rC, Duration 3 weeks) Concentration of PGR ( 1 mg/l BAP) Final pH of Zarrouk’s Media Zarrouk’s Media+PGR Biomass (gm/ 250 ml)Frequency of biomass Growth (%) Color9.60 0.53 70 green, few clumps, no contamination Zarrouk’s Media0.56 75 Dark green, thick few clumps, no contamination 9.50 Agitation Zarrouk’s Media0.59 80 Dark green, thick few clumps, no contamination 9.60 Without Agitation Zarrouk’s media0.55 73 green, thick few clumps, no contamination, thin film of cells on flask wall 9.40 Figure-1 (A-G)A. S. Platensis on Zarrouk’s agar media, B. Microscopic view of S. Platensis, C. S. Platensis in CFTRI Media, D.S. Platensis in Bangladesh Media, E. S. Platensis in RM-6 Media, F. S. Platensis in OFERR Media, G. S. Platensis in A-5 Media International Research Journal of Biological Sciences _______ Vol. 1(6), 49-56, October (2012) International Science Congress Association Biomass production of Biomass production of Biomass production of  \n  \r    \n   \r      (Temperature 25 \rC, Duration 3 weeks)      ! "   !    (Temperature 25 \rC, Duration 3 weeks)   #\r$\r% %\r&  (Temperature 25 \rC, Duration 3 weeks)   International Research Journal of Biological Sciences _______ _________________________________________ International Science Congress Association Figure-2 Biomass production of Spirulina pletensis different temperature Figure-3 Biomass production of Spirulina pletensis different light intensity Figure-4 Biomass production of Spirulina pletensis different carbon and nitrogen source  \n   \r    \n  \r   \n  \r    \n   \r    ""  Microorganism (Spirulina pletensis (Temperature 25 \rC, Duration 3 weeks) Initial pH 8 of Zarrouk’s Media   ' $()%%*+,\r- "   !    !  !  !  !  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Biological Sci. 53 different carbon and nitrogen source  \n   International Research Journal of Biological Sciences _______ Vol. 1(6), 49-56, October (2012) International Science Congress Association Biomass production of Biomass production of $ "$  Microorganism ( (Temperature 25 \rC, Duration 3 weeks)   " '#./ ,\r-  -\r\n ,\r- Frequency of Biomass growth (%) International Research Journal of Biological Sciences _______ _________________________________________ International Science Congress Association Figure-5 Biomass production of Spirulina pletensis in different pHFigure-6 Biomass production of Spirulina pletensis different medium "$ $$ $ "" Microorganism ( Spirulina pletensis (Temperature 25 \rC, Duration 3 weeks)   ' $()%%*+,\r-  -\r\n ,\r- ., 0  ,\r- 1 0 ,\r- !''.. ,\r- '%\r2\r 3(4 5%6 \n7 _________________________________________ ISSN 2278-3202 I. Res. J. Biological Sci. 54 $ "" '%\r2\r 3(4 5%6 \n7 International Research Journal of Biological Sciences _______ Vol. 1(6), 49-56, October (2012) International Science Congress Association Biomass production of Spirulina pletensis Improved Vital Biotech (2012) Media for production of pletensis: Culture development and maintenance: pletensis was grown in i mproved vital biotech (2012) Media. Firstly, we have transferred our culture in biotech (2012) Media. Cultures were incubated in a culture room at temperature of 25 C. During the process of growth the flask was shaken 3 to 4 time per day and colour of vital biotech (2012) Media was Dark green and no contamination, thin film of cells on flask wall. Preparation of Improved vital biotech (2012) media as following step: Firstly we had taken sterilized 500ml conical flask. Then we had ta ken 250ml D/W in flask. After that mix properly all components of improved vital biotech media. Result described in table-7. Table-7 Improved Vital Biotech (2012) Media for production of pletensisIngredients Sodium bicarbonate Sodium chloride Urea Sodium nitrate Potassium sulphate Magnesium sulphate Ferrous sulphate Potassium di hydrogen phosphate )%%*8 \r-9:5. )%%*8\r-  Microorganism ( (Temperature 25 \rC, Duration 3 weeks) Concentration of PGR ( 1 mg/l BAP)   International Research Journal of Biological Sciences _______ _________________________________________ International Science Congress Association Figure-7 Spirulina pletensis in different chemical and physical environment (2012) Media for production of S. maintenance: Spirulina mproved vital biotech (2012) Media. Firstly, we have transferred our culture in improved vital biotech (2012) Media. Cultures were incubated in a culture C. During the process of growth the colour of improved Dark green and no contamination, thin film of cells on flask wall. Preparation of Improved vital biotech (2012) media as Firstly we had taken sterilized 500ml conical ken 250ml D/W in flask. After that mix vital biotech (2012) (2012) Media for production of S. gm/lit 8.0 5.0 0.2 2.5 0.5 0.16 0.05 0.052 Preparation of inoculum: Inoculum preparation for culture maintenance taken well developed biomass concentration of Spirulina culture, which has inoculated before 20 to 25 days in i mproved vital biotech (2012) Media. Filtration: Cells were collected by filtration using filter paper 8mm pore size (s creen printing paper). Washing: Cells were washed with buffer solution (pH 7), diluted to known volume and processed for further inoculation. Dry weight measurement: For dry weight measurement homogenous suspension of known quantity of were filtered through screen- printing paper and oven dried at 75ºC for 2 to 6 hours. The dried filter paper containing biomass were cooled and weighted. The di initial and final weight were taken as the dry weight of biomass. The dry weights were expressed in terms of 0.82 gm/liter. Final pH was observed of Improved VITAL BIOTECH (2012) Media is pH 10. We have observed that biotech (2012) Media is good media for biomass production of Spirulina platensis. ConclusionThe genus spirulina , is the most important commercially cultivated cynobacterium, due to its high nutritional value, chemical composition and safety of it )%%*8\r- 1    )%%*8 \r- 6 \n 1     )%%*8\r-  Microorganism ( Spirulina pletensis (Temperature 25 \rC, Duration 3 weeks) Concentration of PGR ( 1 mg/l BAP)   ' $()%%*+,\r- _________________________________________ ISSN 2278-3202 I. Res. J. Biological Sci. 55 in different chemical and physical environment Inoculum preparation for culture maintenance taken well developed biomass concentration of culture, which has inoculated before 20 to 25 days in mproved vital biotech (2012) Media. by filtration using filter paper creen printing paper). Cells were washed with buffer solution (pH 7), diluted to known volume and processed for further inoculation. For dry weight measurement homogenous suspension of known quantity of Spirulina sample printing paper and oven dried at 75ºC for 2 to 6 hours. The dried filter paper containing Spirulina biomass were cooled and weighted. The di fference between the initial and final weight were taken as the dry weight of Spirulina biomass. The dry weights were expressed in terms of 0.82 was observed of Improved VITAL BIOTECH (2012) Media is pH 10. We have observed that improved vital (2012) Media is good media for biomass production of , is the most important commercially cultivated cynobacterium, due to its high nutritional value, chemical composition and safety of it s biomass for human 6 \n 1     )%%*8\r-  International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 1(6), 49-56, October (2012) I. Res. J. Biological Sci. International Science Congress Association 56 consumption. It is cultivated on a large scale as a monoculture in intensive outdoor cultivation system. Standardization of Spirulina in different media was summarized maximum growth noticed in Zarrouk’s media. As it is after the treatment of different pH the best growth resulted in pH 11. Aeration effect was important for Spirulina cultivation. Aeration agitates the growth medium and this gives homogenous distribution of Spirulina filaments throughout the growth vessel for adequate exposure to illumination uniformly and removes some inhibitory substances produced such as carbon dioxide. This phenomenon is similar in outdoor cultivation of Spirulina strain. Aeration is essential for the cultivation of the Spirulina pletensisit is also noted that continuous mixing of the culture medium is required to prevent cell sinking and thermal stratification, maintain even nutrient distribution, and to remove excess oxygen. The biomass production of Spirulina species was lower in light intensity (435lux) and higher when the growth was illuminated in 1300 lux. Spirulina inoculated in improved VITAL BIOTECH medium was survived and growth was flourished achieving dry weight of 0.82 dw/L on 20th day of cultivation. 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