WO2006018668A1 - Procede economique et efficace de production en masse de spiruline - Google Patents

Procede economique et efficace de production en masse de spiruline Download PDF

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Publication number
WO2006018668A1
WO2006018668A1 PCT/IB2004/002643 IB2004002643W WO2006018668A1 WO 2006018668 A1 WO2006018668 A1 WO 2006018668A1 IB 2004002643 W IB2004002643 W IB 2004002643W WO 2006018668 A1 WO2006018668 A1 WO 2006018668A1
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WIPO (PCT)
Prior art keywords
ranging
spirulina
culture
seawater
composition
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PCT/IB2004/002643
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English (en)
Inventor
Mallappa Mahadeva Swamy
Kotamballi Nagendra Murthy Chidambara Murthy
Gokare Aswathanarayana Ravishankar
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Council Of Scientific And Industrial Research
Department Of Biotechnology
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Priority to AU2004322412A priority Critical patent/AU2004322412B2/en
Priority to PCT/IB2004/002643 priority patent/WO2006018668A1/fr
Publication of WO2006018668A1 publication Critical patent/WO2006018668A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor

Definitions

  • the invention relates to a seawater based medium composition for Spirulina production.
  • Spirulina are blue-green algae belonging to the phylum: Cyanophyta, class Cyanophyceae, order: Nostocales, family: Oscillatoriaceae, genus: Spirulina or Arthrospira.
  • Spirulina is one of the most comprehensive sources of nutrition has known to man.
  • Algae are rich in gamma linolenic acid (GLA), linoleic and arachidonic acids. They are also rich in iron, protein, essential amino acids, vitamins, minerals and chlorophyll.
  • GLA gamma linolenic acid
  • S. fusiformis Bourrelly P. 1970. Les alguesdiandianes ou cyanophyces, in "Les Algues d'eauless, Tome III, Editions N. Boube).
  • Spirulina is a nutrient-rich (Table 1, Becker E. W. and Venkataraman L. V., 1982, Biotechnology and exploitation of algae - The Indian Approach, Publication "German Agency for technical co-operation,, D-6236, Eschborn 1, Federal Republic of Germany, Druckerei Gneiting GmBH Filmsatz+Druck, T ⁇ bingen) microalga and processed as dry powder. It can be consumed as powder or as tablets for availing health benefits. The powder may also be added to many dishes to enhance the protein and vitamin content. A variety of recipes are available to prepare Spirulina based dishes. It can also be added to fruit or vegetable juice.
  • Spirulina powder is sold with recipes which, include pasta, whole wheat Spirulina bread, Spirulina drink, scones, whole meat biscuits, soups, pastalina, fermented products like Dihe, Tofu, Bread spreads, sources, salad dressings, curries, herb filling and deserts. (United States Patent Application- 20030017558 pham, quoc kiet; et al.
  • GLA gamma linolineic acid
  • GLA United States Patent Application-20030017558 pham, quoc kiet; et al. -Method for mixotrophic culture of Spirulina for producing a biomass rich in omega w6 polyunsaturated fatty acids and/or in sulpholipids).
  • Glycolipids extracted from Spirulina have been found to combat the AIDS virus (Boyd,
  • GLA has been found to have a positive effect in the treatment of arthritis (Belch, et al, 1988), and premenstrual
  • Spirulina which contains nutritional and highly valuable bioactive compound, can help to many health problems by one or the other mechanism. Researches done on Spirulina have revealed that it can be beneficial in following cases,
  • Beta-carotene is one of the most well known anti-cancer substances. It is the precursor of vitamin A. Since beta-carotene is present in very high amount in Spirulina, a diet comprising of Spirulina will reduce cancer risks.
  • Table. 1 Composition of Spray dried Spirulina (Percentage on dried weight)
  • Spirulina has the highest protein content (60-70%) of any natural food. It has no hard cellulose in its cell walls, being composed of soft mucopolysaccharides. This ensures its protein is easily digested and assimilated in the human body. It is 85 to 95% digestible. Spirulina protein contains all essential amino acids in adequate quantities (Table-2, Becker E. W. and Venkataraman L. V., 1982, Biotechnology and exploitation of algae - The Indian Approach, Publication "German Agency for technical co- operation,, D-6236, Eschborn 1, Federal Republic of Germany, Druckerei Gneiting GmBH Filmsatz+Druck, T ⁇ bingen), which is comparable with any other vegetables and meat. Table - 2. Amino acids composition of Spirulina (all in dry weights)
  • the vitamin content of Spirulina reflects another important benefit as a human food, representing a rich natural source of vitamins.
  • a ten-gram serving of Spirulina supplies a rich profile of vitamins we need. It is the richest source of beta-carotene (precursor of Recently it has been experimentally proven that aqueous extract of Spirulina is helpful in suppressing AIDS where it has been linked to the inhibition of HIV-I replication (Ayehunie et al, 1998, J. of AIDS illumination Retroviral, Vol. 18: 7-12).
  • Spirulina has also been implicated in immuno-modulating activities (Hirayashi et al, 2002, International Immunopharmacology, Vol. 2: 423-434) and against arthritis (Remerez et al, 2002, Mediators Inflamm. Vol. 11 : 75-79).
  • Venkataraman L. V. Mass production of the blue- green algae Spirulina: an overview, Biomass, Vol. 15: 233 - 247) wherein nutrient medium was further modified to contain a very simple nutrient mixture composed of the following commercial grade constituents: Sodium bicarbonate 4.Og, Urea (CO(NH 2 ) 2 ) 0.5g, Sodium chloride 1.0 g, Potassium sulphate 1.0 g, Potassium hydrogen phosphate 0.5g, Magnesium sulphate 0.2Og, and water to make up I L where though no additional ferrous sulphate in the medium was used, the resultant biomass contained 50 mg iron accumulation per lOOg of dry weight of Spirulina which probably resulted from traces of ferrous sulphate contamination in the commercial grade chemicals.
  • Vitamin A (beta carotene) 23000 IU 5000IU
  • Vitamin B2 (riboflavin) ' 0.35 mg 1.7 mg
  • Vitamin B 3 (niacin) 1.46 mg 20 mg V Viittaammiinn B B66 ( (ppyyrriiddooxxiinnee)) 8 800 m meegg 2 mg
  • Pigments help synthesis of many enzymes necessary for regulating the body's metabolism.
  • Phycocyanin and phycocyanobilin from Spirulina has been linked to scavenging of peroxinitrite free radicals leading to protection against oxidative damage to Deoxyribonucleic acid (DNA) (Bhat and Madyastha, 2001, Biochem. Biophys. Res. Commun. Vol. 285: 262-266).
  • Chlorophyll contains a magnesium ion at its core, giving it a green colour, and hemoglobin contains iron, giving it a red colour.
  • Spirulina for anemic patients could be due to the conversion of chlorophyll into hemoglobin, as well as the high bioavailbe iron content of Spirulina (Puyfoulhoux et al, 2001, J. Agric. Food Chem. Vol. 49: 1625-1629). Apart from these, Spirulina contains several useful enzymes in adequate quantities. Superoxide dismutase (SOD) enzyme activity ranging from 10,000 to 37,000 EU per ten grams in Spirulina powder is linked to its very high free radical scavenging effects in human body, imparting anti-cancerous property to Spirulina (Babu, 1995, Nutrition and Cancer Vol.
  • SOD superoxide dismutase
  • the main object of the present invention is to provide a seawater based medium composition for Spirulina production.
  • Another object of the present invention is to obtain growth of Spirulina in synthetic or natural seawater. Another object of the present invention is to produce Spirulina with most essential constituents comparable to the culture growth using Zarrouk's or CFTRI medium. Summary of the Present Invention
  • the present invention relates to an economical and efficient method for mass production of spirulina using seawater-based medium composition of pH ranging between 6.5 and 8.0 comprising sodium bicarbonate of concentration ranging between 1.2 to 3.0 % w/v, nitrogen of concentration ranging between 0.1 to 0.3% w/v, phosphorus of concentration ranging between 0.1 to 0.3% w/v, potassium of concentration ranging between 0.1 to 0.3% w/v seawater and composition thereof.
  • the present invention relates to an economical and efficient method for mass production of spirulina using seawater-based medium composition of pH ranging between 6.5 and 8.0 comprising sodium bicarbonate of concentration ranging between 1.2 to 3.0 % w/v, nitrogen of concentration ranging between 0.1 to 0.3% w/v, phosphorus of concentration ranging between 0.1 to 0.3% w/v, potassium of concentration ranging between 0.1 to 0.3% w/v seawater and composition thereof.
  • An economical and efficient method for mass production of spirulina using seawater- based medium composition of pH ranging between 6.5 and 8.0 comprising sodium bicarbonate of concentration ranging between 1.2 to 3.0 % w/v, nitrogen of concentration ranging between 0.1 to 0.3% w/v, phosphorus of concentration ranging between 0.1 to 0.3% w/v, potassium of concentration ranging between 0.1 to 0.3% w/v seawater, said method comprising steps of: o growing the spirulina in agar slants using standard Zarrouk's medium at temperature ranging between 25 to 35 0 C, illumination ranging between 1000 to 2000 lux, with photoperiod of 12 to 16 hours per day for total cultivation period ranging between 25 to 40 days to obtain a culture, o Transferring the culture to seawater-based medium composition with initial optical density of about 0.1 at about 560 nm, o growing the culture of step (b) at temperature ranging between 25 to
  • step (c) is about 6 to 12 days.
  • the culturing is in carboy.
  • the method using seawater for culturing makes the method economical.
  • seawater can be both natural and synthetic.
  • the present invention also relates to a seawater based medium composition useful for mass production of spirulina, said composition comprising sodium bicarbonate of concentration ranging between 1.2 to 3.0 % w/v, nitrogen of concentration ranging between 0.1 to 0.3% w/v, phosphorus of concentration ranging between 0.1 to 0.3% w/v, potassium of concentration ranging between 0.1 to 0.3% w/v in seawater.
  • the composition is of pH ranging between 6.5 to 8.0.
  • Seawater is one of the widely available natural resource, which can be utilized for various purposes such as , cultivation of marine organisms like, seaweeds, fish and marine algae. Seawater is rich in various nutrients, which supports algal life (eg. Dunaliella salina, Spirulina). Since seawater contains less sodium bicarbonate, sodium nitrate and phosphorous, therefore seawater supplementation with these nutrient can serve as complete medium and economic source of Spirulina cultivation. This facilitates cultivation of Spirulina in coastal region using seawater.
  • the present invention provides a seawater based medium composition for Spirulina production.
  • a seawater based medium composition for Spirulina production which comprises of a growing the stock cell culture of Spirulina platensis in agar slants using standard Zarrouk's medium, at 25-35° C under 1000 - 2000 lux illumination for a photoperiod of 12-14h per day for a total cultivation period of one month, transferring the cells from stock culture to liquid medium in carboy where the chemical composition of the synthetic sea water / sea water supplemented medium containing Sodium bicarbonate 15.0 to 32.0, Sodium nitrate 2.0 to 9.0 , Sodium chloride 25 to 62, Magnesium sulphate 5.0 to 18.0 , Magnesium chloride 4.0 to 15.0, Potassium chloride 0.5to 3.4, Boric acid 0.01 to 0.10, Suphala 0.4 to 3.0 values are gm per liter basis of the total salt and the volume made up with domestic water, ensuring
  • the stock culture of Spirulina platensis ⁇ selected from a group of stains grown in agar slants using standard Zarrouk's medium, at 25-35° C under 1000 - 2000 lux illumination for a photoperiod of 12-16h per day for a total cultivation period of one month.
  • the cultures are transferred to carboy where the chemical composition of the medium includes Sodium bicarbonate 15.0 to 32.0, Sodium nitrate 2.0 to 9.0, and Potassium phosphate 0.1 to 2 g/1 in sea water.
  • the cultures are transferred from stock culture to liquid medium in carboy containing sea water supplemented with Sodium bicarbonate 10.0g, Suphala (Agricultural fertilizer N:P: K:: 15: 15: 15)0.5g ,Urea 0.2g in IL of sea water, ensuring the initial optical density of 0.1 at 560nm and growing Spirulina cells at 25-35° C under 20-30 Klux illumination for a photoperiod of 12-14h per day for a total cultivation period of about one week or till the culture reaches 1.0 optical density at 560 nm.
  • the cultures of Spirulina platensis from carboy are transferred to open cement raceway tank of conventional type filled with liquid medium of chemical composition involving Sodium bicarbonate 15.0 to 32.0, Suphala (NPK 15:15: 15), l .Og/1 , Sodium chloride 2.5 to 30, Magnesium sulphate 4.0 to 18.0, Magnesium chloride 4.0 to 15.0, Potassium chloride 0.5to 3.4, Boric acid 0.01 to 0.10, Suphala 0.4 to 3.0 and the volume made up with domestic water and filling the tank up to 12-20 cm depth, ensuring the initial optical density of 0.1 at 560nm and growing Spirulina cells at 25-35° C, and agitating the culture with a conventional paddle wheel at a speed of 8-12 rotation per minute under 30-45 K.Lux illumination for a photoperiod of 8-1Oh per day for a total cultivation period of about one week or till the culture reaches 2.0 optical density at 560 nm.
  • liquid medium of chemical composition
  • the harvesting and drying of the biomass was done as per the standard industrial procedure followed and where the biomass is analyzed for the nutritional composition (table 2).
  • the biomass is analyzed for the nutritional composition (table 2).
  • Example 1 Among the different strains of Spirulina platensis, the strain SP-6 was selected as a best performing strain under various light, temperature and nutrient conditions and preserved for future use.
  • the nutrient medium used for maintaining the stock culture namely the Zarrouk's culture medium, was prepared using the following chemicals:
  • the chemicals are dissolved in distilled water and the final volume is made up to one litre, the pH is adjusted to about 8.5, after which 10 grams of agar is added, and agar slants preparation and Spirulina culture inoculations are done as per standard microbiological methods. Cultures are maintained at a temperature ranging from 20 to 22 0 C and an illumination cycle of about 16 h of white light of 1000 - 2000 lux and 8 h in dark is maintained. The cells are allowed to grow for a period of 25-40 days.
  • Example 2 For indoor liquid inoculum development, the Spirulina cells cultured in agar slants grown as described in example 1 are taken by aseptically scraping the surface of the medium with a spatula and the cells are suspend in sea water (obtained from coastal region of Mangalore, Karnataka State). However, studies were further carried with synthetic seawater prepared as per the composition below medium present in glass carboys of 5L capacity, where the medium has the following chemical composition: Composition of sea water and synthetic sea water
  • the aforementioned table shows the standard composition of seawater (left column) and also, the final composition of the seawater after incorporating the supplements of the seawater-based medium composition.
  • the compounds grams
  • the suspended cells are incubated at ambient condition with daily 3 to 4 times by manual agitation.
  • the initial optical density of 0.1 at 560nm is ensured and further cell growth is allowed at 25-35°C under 20-30 Klux illumination for a photoperiod of 12-14h per day for a total cultivation period of about one week or till the culture reaches 1.0 optical density at 560 nm. (OD 1.0, which corresponds to 900-1000 mg dry algae/liter).
  • the culture broth of optical density 1.0 is diluted 10 times using synthetic sea medium, and the required level of inoculum is thus developed. Further, the presence of urea and calcium chloride is optional in the final composition of the modified seawater.
  • the cell culture broth developed as described in example 2 is subsequently used to produce large quantity of cell biomass in open conventional raceway ponds.
  • the basic design we suggest for mass cultivation of Spirulina consists of oblong, shallow raceway type pond / tank stirred with paddle wheel. Commercial pond area varies between 5 to 5000 m 2 .
  • the Spirulina cultures from several carboys is transferred to 5 m 2 tank with the addition of 0.90 m 3 water fortified with the nutrients of medium the composition of which as above:
  • Optimum condition a. Light (Kilolux) 35-45 b. Temperature 25-35 0 C c. Inoculum size of OD 0.1 d. Nutrient new seawater medium e. Culture depth (cms) 18-20 f. Mixing/agitation using
  • Proximate composition of Spirulina cultivated in different media (in % value).
  • Harvesting the Spirulina cells was done by gravity filter or any other available conventional method. Harvested biomass was washed with 0.01% hydrochloric acid in a solution in order to remove surface coated NaHCO 3 and bound minerals. Moreover, the salt concentration of the rinsing solution, which is similar to one of the culture medium, permits to avoid a breaking of the cellular membranes due to osmotic pressure. The biomass is finally rinsed with tap water. The harvested Spirulina biomass can be lyophilized or sprayed, preferably immediately, in order to avoid a secondary contamination. Spirulina may also be dried by any other standard dying method such as spray-drying, vacuum-drying, cross-flow drying, sun-drying etc.

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Abstract

La présente invention concerne un procédé économique et efficace de production en masse de spiruline utilisant une composition d'un milieu à base d'eau de mer avec un pH compris entre 6,5 et 8,0, qui contient du bicarbonate de sodium ayant une concentration comprise entre 1,2 et 3,0 % p/v, de l'azote dans des concentrations comprises entre 0,1 et 0,3 % p/v, du phosphore dans des concentrations comprises entre 0,1 et 0,3 p/v, du potassium dans des concentrations comprises entre 0,1 et 0,3 % p/v, de l'eau de mer, ainsi qu'une composition correspondante.
PCT/IB2004/002643 2004-08-13 2004-08-13 Procede economique et efficace de production en masse de spiruline WO2006018668A1 (fr)

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AU2004322412A AU2004322412B2 (en) 2004-08-13 2004-08-13 An economical and efficient method for mass production of spirulina
PCT/IB2004/002643 WO2006018668A1 (fr) 2004-08-13 2004-08-13 Procede economique et efficace de production en masse de spiruline

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2176396A2 (fr) * 2007-06-18 2010-04-21 Choudhary, Vidhi Algues jaunes dorées et leur procédé de production
US20120021496A1 (en) * 2010-07-23 2012-01-26 Eulgi University Industry Academy Cooperation Foundation Method for circulatory cultivating photosynthetic microalgae
FR3019832A1 (fr) * 2014-04-11 2015-10-16 Capodano Marie-Gabrielle Puppo Dispositif de realisation d'un melange de spiruline a l'eau de mer en culture vivante
CN105754914A (zh) * 2016-05-18 2016-07-13 青岛农业大学 一种用于海水螺旋藻培养的培养液
WO2017064373A1 (fr) * 2015-10-15 2017-04-20 Puppo Capodano Marie-Gabrielle Dispositif de realisation d'un melange de spiruline a l'eau de mer en culture vivante

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1138757A1 (fr) * 1999-09-29 2001-10-04 Micro Gaia Co., Ltd. Procede de mise en culture d'algues permettant de produire des pigments phototropes, des acides gras fortement insatures ou des polysaccharides a forte concentration
US20030017558A1 (en) * 1997-09-19 2003-01-23 Quoc Kiet Pham Method for mixotrophic culture of spirulinas for producing a biomass rich in omega w6 polyunsaturated fatty acids and/or in sulpholipids

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030017558A1 (en) * 1997-09-19 2003-01-23 Quoc Kiet Pham Method for mixotrophic culture of spirulinas for producing a biomass rich in omega w6 polyunsaturated fatty acids and/or in sulpholipids
EP1138757A1 (fr) * 1999-09-29 2001-10-04 Micro Gaia Co., Ltd. Procede de mise en culture d'algues permettant de produire des pigments phototropes, des acides gras fortement insatures ou des polysaccharides a forte concentration

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COSTA JORGE ALBERTO VIEIRA ET AL: "Improving Spirulina platensis biomass yield using a fed-batch process.", BIORESOURCE TECHNOLOGY. MAY 2004, vol. 92, no. 3, May 2004 (2004-05-01), pages 237 - 241, XP002306500, ISSN: 0960-8524 *
FAUCHER O ET AL: "Utilization of seawater-urea as a culture medium for Spirulina maxima.", June 1979, CANADIAN JOURNAL OF MICROBIOLOGY. JUN 1979, VOL. 25, NR. 6, PAGE(S) 752 - 759, ISSN: 0008-4166, XP009040238 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2176396A2 (fr) * 2007-06-18 2010-04-21 Choudhary, Vidhi Algues jaunes dorées et leur procédé de production
EP2176396A4 (fr) * 2007-06-18 2010-12-29 Choudhary Vidhi Algues jaunes dorées et leur procédé de production
US20120021496A1 (en) * 2010-07-23 2012-01-26 Eulgi University Industry Academy Cooperation Foundation Method for circulatory cultivating photosynthetic microalgae
FR3019832A1 (fr) * 2014-04-11 2015-10-16 Capodano Marie-Gabrielle Puppo Dispositif de realisation d'un melange de spiruline a l'eau de mer en culture vivante
WO2017064373A1 (fr) * 2015-10-15 2017-04-20 Puppo Capodano Marie-Gabrielle Dispositif de realisation d'un melange de spiruline a l'eau de mer en culture vivante
CN105754914A (zh) * 2016-05-18 2016-07-13 青岛农业大学 一种用于海水螺旋藻培养的培养液

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