new record of potential cyanobacteria from indian region

Philippine Journal of Science141 (1): 57-66, June 2012ISSN 0031 - 7683Date Received: 26 Oct 2010

Key Words: Biodiversity hot spots, Cyanobacteria, Indo-Burma, North-East India, Repository, Value addition

*Corresponding author: [email protected]

57

Ojit Singh K, Gunapati Oinam, and Tiwari ON*

New Record of Potential Cyanobacteria fromIndian Region Falling Indo-Burma Biodiversity Hotspots

(North-East Region of India) and PartialCharacterization for Value Additions

Microbial Bioprospecting Laboratory, Microbial Resources DivsionInstitute of Bioresources and Sustainable Development

(An autonomous Institute under the DBT, Gov't. of India)Takyelpat, Imphal-795001, Manipur, India

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. They appeared to be a rich source for many useful products and are known to produce a number of bioactive compounds. The North-east region of India is a genetic treasure house of plant, animal, and microbial resources. In this study, two-hundred sixty (260) unialgal cyanobacterial isolates from Indian region falling Indo-Burma biodiversity hotspots were characterized and primarily screened. Ten (10) isolates from six genera viz-a-viz, Anabaena (03), Nostoc (01), Phormidium (03), Plectonema (01), Lyngbya (01), and Microchaete (01) were found to be useful for industrial application. Study show that in the present investigation, Phormidium tenue (Menegh.) Gomont (NEMN138) have showed ammonia content and can be used as biofertilizers. Anabaena fuellebornii Schmidle (NEMN125), Phormidium bohneri Schmidle (NEMN183), Nostoc spongiaeforme Agardh ex Born. et Flah (NEMN131) were found to be useful for production of phycobiliproteins from cyanobacteria which can be beneficial for industrial applications. All ten isolates have been deposited to the fresh water cyanobacterial and microalgal repository of IBSD, Imphal, Manipur, India (National facility created by Department of Biotechnology, Government of India, in 2009 with reference No. BT/PR 11323/PBD/26/171/2008 dated 31- 03-2009) after obtaining accession number.

INTRODUCTIONCyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis (Moore 1981). They appeared to be a rich source for many useful products and are known to produce a number of bioactive compounds (Carmichael 2001; Codd 1997). During the last few decades, cyanobacteria have been described as potentially important sources for vitamins, fuels, fine chemicals and

many other pharmaceutical products (Chacon-de-Popioici 1994; DeVries et al. 1993; Miura et al. 1993).

Cyanobacteria play an important role in maintenance and build- up of soil fertility, consequently increasing rice growth and yield as a natural biofertilizer (Song et al. 2005). At present, cyanobacteria generally remain as potential sources for further investigations as prospective and excellent sources of biologically active constituents produced during primary and especially secondary metabolism (Skulberg 2000).

Singh et al.: Potential Cyanobacteria from North-East Region of India

Philippine Journal of ScienceVol. 141 No. 1, June 2012

58

MATERIALS AND METHODS Ten (10) unialgal cyanobacterial isolates were obtained from the fresh water cyanobacterial repository of IBSD, Imphal, Manipur, India (National facility created by Department of Biotechnology, Government of India in 2009) which were earlier collected during 2007-09 from different habitats of NE region of India falling under Indo-Burma biodiversity hotspots (location of North-east India as shown in Figure 1 and 2). The North-east region of India located between 87°32’E to 97°52’E latitude and 21°34’N to 29°50’N latitude is a genetic treasure house of plant, animal and microbial resources. The region forms a distinctive part of the Indo-Burma biodiversity hotspot which ranks 8th among the 34 biodiversity hotspots of the world and is a prime one among the two identified for the Indian sub-continent. Geographical details were also recorded using Global Positioning System (Garmin eTrex Vista, Taipei County, Taiwan). Cultural studies and microphotography have been recorded with using Trinocular Research Microscope (NIKON Eclipse 80 i; Nikon, Tokyo, Japan) and Carl Zeiss fluorescence microscope, Axio Scope A1 (Carl Zeiss, Gottingen, Germany) followed by taxonomical characterization of pure cultures referring to key (Desikachary 1959; Anagnostidis & Komarek 2005). 15 mL of well homogenized culture are inoculated into the BG-11 (-N and +N) broth media in 250 mL conical flask and kept in the culture room under light/ dark cycle (14h/10h) maintained at 28 ± 2ºC under illumination of 4000-5000 lux light intensity. Cultures were uniformly stirred twice for a few minutes every day. Three replicas of each flask with inoculum were maintained for zero day, 15th and 30th days analysis. Cells were harvested by centrifugation (refrigerated Eppendorf centrifuge 5430R) for the estimation of chlorophyll-a, total soluble protein, ammonia excretion, carbohydrates, carotenoids and phycobiliproteins and absorbance was measured using spectrophotometer (Shimadzu spectrophotometer, UV-1800). The above parameters were conducted following the mentioned below methods:

Chlorophyll-a (McKinney 1941); total soluble proteins (Herbert et al. 1971); ammonia excretion (Solarzano 1969); carotenoids (Jensen 1978); carbohydrates (Spiro 1966); and phycobiliproteins (Bennett & Bogorad 1973).

Statistical analysisData obtained was subjected to analysis if variance and least significant difference (LSD) at 5% were tested to separate the means using AgRes software statistical package.

Primary metabolites have been defined as low molecular weight compounds that are necessary for growth (Staley & Stanley 1986). They include amino acids, nucleotides, coenzymes, organic acids and vitamins, as well, as intermediates in the bio-synthetic pathways of these compounds. Among microorganisms, cyanobacteria are rapidly proving to be an extremely important source of biologically active secondary metabolites with potential benefits against human disease. Cyanobacteria are one of the potential organisms, which are useful to mankind in various ways. (Mitsui et al. 1981; Prabhaharan and Subramanian 1995; Gustafson et al. 1989; Sundararaman et al. 1996; Subramanian & Uma 1996). Spirulina is used as food supplement because of its excellent nutrient composition and digestibility. It has high protein content (60 - 70%), 20% carbohydrate, 5% lipids, 7% minerals and 6% moisture. It is also a rich source of beta-carotene, thiamine and riboflavin and is one of the richest sources of vitamin B12. It is commercially available in the market in the form of powder, granules or flakes and as tablets and capsules.

A variety of fine chemicals such as pigments, vitamins and enzymes with varied applications can be obtained on a commercially viable scale from cyanobacteria. A number of cyanobacteria are rich in vitamins and many can excrete them into the surrounding environment (Borowitzka 1988). The carotenoids and phycobiliproteins, characteristic of cyanobacteria have high commercial value. They are used as natural food colourants, as food additives to enhance the colour of the flesh of Salmonid fish and to improve the health and fertility of cattle (Emodi 1978). Feed grade Phormidium valderianum is an excellent source of phycocyanin, a blue natural colorant useful as a phycofluor in diagnostics. Enzymes such as chitinase, L-asparaginase, L-glutaminase, amylase, protease, lipase, cellulase, urease and superoxide-dismutase have been reported from cyanobacteria (Wikstrom 1997). Photoproduction of ammonia and amino acids by cyanobacteria has also been described (Hall and Grassi 1989). Cyanobacteria in general and marine forms in particular are one of the richest sources of known and novel bioactive compounds including toxins with wide pharmaceutical applications. Gustafson et al. (1989) reported anti-HIV activity of marine cyanobacterial compounds from Lyngbya lagerheimii and Phormidium tenue.

The aim of the present investigation is to screen and select the most promising cyanobacteria. Preliminary screening was done based on their biochemical components such as total sugars, total soluble proteins and pigment composition.



59

RESULTS AND DISCUSSIONTwo hundred and sixty unialgal cyanobacterial isolates were primarily characterized and ten isolates were found at par with already commercialized particularly for industrial applications were shown in Table 1. All these ten isolates were first time isolated, purified from fresh water habitats having acidic properties (pH 5.0 - 6.5) from North-east region of India falling under Indo-Burma biodiversity hotspots, earlier unexplored due to its remote locations. Each and every isolates having its own uniqueness and again deposited to fresh water cyanobacterial and microalgal repository of IBSD, Imphal, Manipur, India (National facility created by Department of Biotechnology, Government of India in 2009). Available chlorophyll-a, total soluble proteins and carotenoids were presented in Table 2.

Chlorophyll-a was recorded maximum during 30th day of growth. Plectonema nostocorum Bornet ex Gomont, Accession No: NEMN158 (5.72 μgmL-1) showed highest chlorophyll-a content with comparison to others on 30th day of growth (Table 2). Total soluble protein was also recorded maximum during 30th day growth. Anabaena variabilis Kutzing ex Born. et Flah, Accession No: NEMN69 (106.00 μgmL-1) showed highest soluble protein content with comparison to others on 30th day of growth (Table 2). Carotenoid content was found maximum during 30th day growth. Anabaena fuellebornii Schmidle, Accession No: NEMN125 (59.29 μgmL-1) showed highest carotenoids content with comparison to others on 30th

day of growth (Table 2). Released ammonia excretion and available carbohydrate are presented in table 3. Ammonia excretion (144.75 μgmL- 1) and carbohydrate (88.00 μgmL-1) were recorded maximum during 30thday growth in non-heterocystous filamentous cyanobacterium, Phormidium tenue (Menegh) Gomont, Accession No: NEMN138 (Table 3). Phycobiliproteins [phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC)] are presented in Table 4.

Anabaena fuellebornii Schmidle, Accession No: NEMN125 (152.34 μgmL-1) produced maximum content of phycoerythrin after 15th day of growth than the other investigated isolates. Phormidium bohneri Schmidle Accession No: NEMN183 and Nostoc spongiaeforme Agardh ex Born. et Flah Accession No: NEMN131 produced maximum amount of phycocyanin (206.14 μgmL-

1) and allophycocyanin (84.73 μgmL-1) respectively on 15th day of growth (Table 4). Since these isolates were found to be potent isolates, they can be commercialized. They can also be further genetically modified for better yield. Our findings reported in this paper are comparable to the previous workers (Cohen 1986; Dainippon Patent 1980; Dainippon Patent 1981; Borowitzka 1994). Apart from the use of phycobiliproteins as food-grade dyes, they ere also used as tools for basic research and medical diagnostics. Phycocyanin, the major phycobiliproteins also exhibited anti-cancer activity, stimulation of immune system and ability to treat ulcers and haemmorrhoidal bleeding. A commercial process of phycocyanin production from open

Figure1. Map of India Figure 2. Map showing North-east region of India where algal samples were collected.



60

scale cultivation of a marine cyanobacterium Phormidium valderianum BDU 30501 was developed (Sekar & Subramanian 1998). A large array of natural products of economic potential may be produced from cyanobacteria. These also represent an attractive source of natural

pigments such as phycobiliproteins, amounting to about 20 percent of the total dry weight vis-à-vis phycocyanin and phycoerythrin and allo-phycocyanin (Borowitzka 1988), and carotenoids (Walsh et al. 1997).

Table 1. Cultural studies and taxonomical characterization of selected cyanobacterial isolates from North-East Region of India.

Name of the isolates Habitat with geographical details of the isolates Brief cultures/ taxonomical studies

Anabaena naviculoides FristchAccession No: NEMN61FWCR

Loktak Lake, Sendra, Bishnupur, Manipur, IndiaAltitude:772mLatitude/ Longitude: N24º30’51.8” E093º47’36.0”

Thallus is brownish colour, thin membrane like biomass, submerged, filaments straight or bent, cells longer than broad with 5.5-6 µm broad, heterocyst almost spherical with 3.2-4 µm broad

Anabaena variabilis Kutzing ex Born. et FlahAccession No: NEMN69FWCR

Loktak Lake, Sendra, Bishnupur, Manipur, IndiaAltitude:772mLatitude/ Longitude: N24º30’51.8” E093º47’36.0”

Thallus brown colour, submerged, compact biomass, filaments slightly constricted at cross-wall, end cells conical, barrel-shaped cells with 2-3 µm long , sometimes with gas vacuoles, heterocysts spherical or oval with 6-7 µm broad, hyaline sheath

Anabaena fuellebornii SchmidleAccession No: NEMN125; FWCR

River Bank, Churachandpur, Manipur, IndiaAltitude: 829mLatitude/Longitude: N24º19’06.4” E093º41’23.5”

Thallus dark brown, submerged, chain of beads, caespitose, filaments coiled or straight, cylindrical cells with 11-12 µm long, heterocyst cylindrical with 17-18 µm long, spores ellipsoidal to oblong cylindrical

Nostoc spongiaeforme Agardh ex Born. et FlahAccession No: NEMN131; FWCR

Foothill, Baruni, Imphal East, Manipur, IndiaAltitude: 785mLatitude/ Longitude: N24º50’40.2” E094º03’26.0”

Thallus dark green, submerged, caespitose biomass, filaments flexuous, cells cylindrical or barrel shaped with 6-7 µm, heterocyst sub-spherical or oblong with 7-8 µm broad and spores oblong, epispore smooth, colourless

Phormidium tenue (Menegh.) GomontAccession No: NEMN138; FWCR

Foothill, Baruni, Imphal East, Manipur, IndiaAltitude: 785mLatitude/ Longitude: N24º50’40.2” E094º03’26.0”

Thallus dark green, floating, cushion like biomass, trichome flexuous with 3-4 µm broad , densely entangled, trichome 3-4 µm broad constricted at the cross-walls, attenuated at the ends, cells longer than broad, end-cell rounded and calyptra absent

Plectonema nostocorum Bornet ex GomontAccession No: NEMN158; FWCR

Lithophilic, Wangkhei, Imphal East, Manipur, IndiaAltitude: 772mLatitude/ Longitude: N24º48’54.2” E093º 56’13.1”

Thallus dark green, initially floating and later submerged, caespitose biomass, filaments irregularly curved, trichome with 9-10 µm broad, false branching, single or geminate, distinctly constricted at the cross-wall and end cell rounded, thin sheath

Phormidium bohneri SchmidleAccession No: NEMN183; FWCR

Muddy Pond, Moreh, Chandel, Manipur, IndiaAltitude: 213mLatitude/Longitude: N24º15’11.1” E094º17’50.5”

Thallus blue-green, half submerged and later floating, floccose (cottony) biomass, cells mostly shorter than broad, round at the tip, trichome with 9-10 µm broad, thin hyaline sheath present

Lyngbya truncicola GhoseAccession No: NEMN184; FWCR

Rice field, Churachandpur, Manipur, IndiaAltitude: 823mLatitude/Longitude: N24º17’12.9” E093º40’49.0”

Thallus dark brown, initially floating and later submerged, thick biomass, membranous, filaments straight, trichome 18-19 µm broad not attenuated towards ends, with finely granular content, apical cells widely rounded, calyptra absent, distinct sheath

Phormidium fragile (Meneghini) GomontAccession No: NETR201; FWCR

Rice field, West Agartala, Tripura, IndiaAltitude: 6mLatitude/Longitude: N23º52’58.7” E091º14’53.4”

Thallus dark green, initially bottom attached and later floating, thin membranous biomass, trichome 4-5 µm broad, hyaline sheath, constriction at the cross-walls, attenuated ends

Microchaete uberrima Carter, N.Accession No: NEMN304; FWCR

Lithophilic, Moreh Chandel, Manipur, IndiaAltitude: 213 mLatitude/ Longitude: N24º15’11.1” E094º17’50.5”

Thallus brown, initially bottom dweller and later floating, thin membranous biomass, filament elongate, quadrate cells with 7-8 µm broad, spherical heterocyst with 7-8 µm broad and present at the tip

Altitude means above sea level



61

Table 3. Released extra-cellular ammonia and total carbohydrate of selected isolates of cyanobacteria.

Name of the isolatesAmmonia Excretion (µgmL-1) Carbohydrates (µgmL-1)

0th day 15th day 30th day 0th day 15th day 30th day

Anabaena naviculoides Fristch 69.00 ± 0.00 85.65 ± 0.00 105.60 ± 0.00 7.00 ± 0.00 15.00 ± 0.00 60.00 ± 0.00

Anabaena variabilis Kutzing ex Born. et Flah 73.00 ± 0.03 88.65 ± 0.00 103.60 ± 0.00 3.00 ± 0.00 29.00 ± 0.01 58.00 ± 0.00

Anabaena fuellebornii Schmidle 71.70 ± 0.00 96.90 ± 0.00 74.40 ± 0.00 9.00 ± 0.04 29.00 ± 0.04 78.00 ± 0.03

Nostoc spongiaeforme Agardh ex Born. et Flah 78.00 ± 0.01 88.50 ± 0.00 69.45 ± 0.00 12.00 ± 0.00 22.00 ± 0.00 39.00 ± 0.00

Phormidium tenue (Menegh.) Gomont 55.50 ± 0.00 69.15 ± 0.00 144.75 ± 0.00 61.00 ± 0.00 73.00 ± 0.02 88.00 ± 0.00

Plectonema nostocorum Bornet ex Gomont 85.35 ± 0.01 102.00 ± 0.01 47.10 ± 0.00 39.00 ± 0.01 48.00 ± 0.00 51.00 ± 0.00

Phormidium bohneri Schmidle 1.50 ± 0.00 48.75 ± 0.01 18.45 ± 0.01 12.00 ± 0.00 42.00 ± 0.02 63.00 ± 0.00

Lyngbya truncicola Ghose 59.70 ± 0.02 83.55 ± 0.00 24.30 ± 0.00 37.00 ± 0.01 56.00 ± 0.02 65.00 ± 0.00

Phormidium fragile (Meneghini) Gomont 45.60 ± 0.03 75.15 ± 0.00 41.70 ± 0.00 19.00 ± 0.01 36.00 ± 0.00 65.00 ± 0.00

Microchaete uberrima Carter, N. 44.70 ± 0.00 69.00 ± 0.00 46.20 ± 0.00 7.00 ± 0.00 27.00 ± 0.02 60.00 ± 0.02

LSD (P≤ 0.05) 0.024 0.010 0.010 0.023 0.028 0.028

All experiments were replicated three times and results are presented as mean ±SD

Table 2. Chlorophyll-a, Total soluble protein, and carotenoids analysis of selected isolates of cyanobacteria from North-East Region of India.

Name of the isolatesChlorophyll-a (µgmL-1) Total soluble protein (µgmL-1) Carotenoids (µgmL-1)

0th day 15th day 30th day 0th day 15th day 30th day 0th day 15th day 30th day

Anabaena naviculoides Fristch 0.50 ± 0.00 1.98 ± 0.01 3.55 ± 0.00 39.00 ± 0.00 58.00 ± 0.03 104.00 ± 0.01 10.48 ± 0.00 25.11 ± 0.00 30.38 ± 0.01

Anabaena variabilis Kutzing ex Born. et Flah 2.90 ± 0.00 4.01 ± 0.00 4.87 ± 0.01 24.00 ± 0.00 77.00 ± 0.03 106.00 ± 0.00 1.92 ± 0.00 17.47 ± 0.01 35.63 ± 0.06

Anabaena fuellebornii Schmidle 1.26 ± 0.01 4.69 ± 0.02 5.57 ± 0.00 34.00 ± 0.00 64.00 ± 0.02 98.00 ± 0.00 18.48 ± 0.00 13.60 ± 0.00 59.29 ± 0.03

Nostoc spongiaeforme Agardh ex Born. et Flah 0.16 ± 0.00 1.76 ± 0.00 3.78 ± 0.01 14.00 ± 0.00 25.00 ± 0.02 50.00 ± 0.00 1.54 ± 0.00 7.80 ± 0.00 24.85 ± 0.00

Phormidium tenue (Menegh.) Gomont 1.82 ± 0.00 3.24 ± 0.04 4.55 ± 0.01 45.00 ± 0.04 72.00 ± 0.03 98.00 ± 0.00 6.05 ± 0.00 14.03 ± 0.00 36.22 ± 0.01

Plectonema nostocorum Bornet ex Gomont 0.16 ± 0.00 1.84 ± 0.00 5.72 ±0.01 4.00 ± 0.00 32.00 ± 0.00 104.00 ±0.01 1.54 ± 0.00 11.9 ± 0.01 25.07 ± 0.00

Phormidium bohneri Schmidle 0.45 ± 0.00 1.72 ± 0.01 4.98 ± 0.01 18.00 ± 0.0 64.00 ± 0.03 98.00 ± 0.01 0.46 ± 0.00 16.08 ± 0.00 27.90 ± 0.01

Lyngbya truncicola Ghose 1.51 ± 0.00 2.43 ± 0.00 3.55 ± 0.01 32.00 ± 0.00 42.00 ± 0.00 74.00 ± 0.00 7.88 ± 0.00 15.65 ± 0.02 29.32 ± 0.05

Phormidium fragile (Meneghini) Gomont 0.22 ± 0.00 2.82 ± 0.00 5.25 ± 0.00 14.00 ± 0.00 43.00 ± 0.00 72.00 ± 0.00 1.11 ± 0.00 12.21 ± 0.00 29.10 ± 0.02

Microchaete uberrima Carter, N. 0.26 ± 0.00 3.64 ± 0.00 4.74 ±0.00 10.00 ± .00 45.00 ± 0.04 101.00 ± 0.00 1.65 ± 0.00 15.72 ± 0.00 31.95 ± 0.01

LSD (P≤ 0.05) 0.008 0.023 0.015 0.020 0.037 0.012 0.009 0.188 0.038




62

Phycoerythrins in particular have applications as natural dyes as well as in fluorescence microscopy. Our findings on ammonia production by cyanobacteria is at par with earlier report (Ramos et al. 1987; Guerrero et al. 1982). They have reported extensively on photosynthetic production of ammonia by cyanobacteria. During growth, they undergo four phases namely, lag, log, stationary and decline. In the present study, chlorophyll-a, total soluble protein, carotenoid, carbohydrates and allophycocyanin content were found to be less during lag phase and more during log phase. This showed that during log phase, metabolic activity will be at the peak. But in case of ammonia, phycoerythrin and phycocyanin, it was found to be more during early stationary growth phase i.e. cells start excreting metabolites during this stage only. The analysis clearly revealed the need for a morpho-physiological and molecular approach for cyanobacterial characterization and their utilization in agriculture and industry. Data generated during present investigations could be useful in understanding of a commercial or biotechnological potential of blue-green algae. Nowadays, scientists are motivated to search for more potential species available in nature for exploiting them in a variety of ways to meet the demands. It needs an extensive screening, which is expected to result in the discovery of better cyanobacterial isolates

of industrial interest. Higher growth rate and nutrient profile of cyanobacteria make them a potentially valuable source of nutrients (Cannell 1989).

Cyanobacteria in general possess all the known phycobiliproteins (phycocyanin, phycoerythrin, phycoerythrocyanin, and all phycocyanin). Among them, phycocyanin and phycoerythrin are commercially valuable. ‘Lina Blue’, a phycocyanin product from Dainippan Ink and chemicals Inc, Japan, is an odourless, non-toxic blue powder and used for colouring candy, ice-cream, dairy products and soft drinks (Cohen 1986). Phycocyanin is also obtained in a water insoluble form from Spirulina and used in eye shadow, eye liner and lipstick preparations (Dainippon Patent 1980). The blue or red chromophores are isolated by enzymatic or acid hydrolysis of the protein to yield more concentrated pigment and used in cosmetics (Dainippon Patent 1981). Phycoerythrin from Spirulina and other cyanobacteria is used as a food colour for products like ice- cream, yoghurt and it could also be used in cosmetics (Borowitzka 1994).

A variety of carotenoids have important commercial uses. Since carotenoids are non-toxic, they are desirable as colouring agents in the food industry as well as vitamin A precursors (Bauernfeind 1981). Furthermore, the flesh, feathers or eggs of fish and birds assume the colour of

Table 4. Investigation of Phycobiliproteins of selected isolates of cyanobacteria from North-East Region of India.

Name of the isolates

Phycobiliproteins (µgmL-1)

PE PE PE PC PC PC APC APC APC

0th day 15th day 30th day 0th day 15th day 30th day 0th day 15th day 30th day

Anabaena naviculoides Fristch 5.58 ± 0.00 123.74 ± 0.00 102.49 ± 0.03 0.77 ± 0.01 69.53 ± 0.01 50.18 ± 0.00 8.42 ± 0.00 19.29 ± 0.01 14.63 ± 0.01

Anabaena variabilis Kutzing ex Born. et Flah 3.22 ± 0.00 69.75 ± 0.01 132.05 ± 0.07 1.50 ± 0.00 47.91 ± 0.00 81.78 ± 0.00 1.70 ± 0.00 50.39 ± 0.00 27.84 ± 0.01

Anabaena fuellebornii Schmidle 1.26 ± 0.00 83.42 ± 0.00 152.34 ± 0.02 2.50 ± 0.00 44.19 ± 0.00 123.77 ± 0.00 0.43 ± 0.00 16.86 ± 0.01 52.59 ± 0.00

Nostoc spongiaeforme Agardh ex Born. et Flah 1.24 ± 0.02 8.63 ± 0.00 57.82 ± 0.02 1.26 ± 0.02 29.43 ± 0.00 47.77 ± 0.10 3.00 ± 0.01 84.73 ± 0.00 26.32 ± 0.01

Phormidium tenue (Menegh.) Gomont

2.51 ± 0.00 3.81 ± 0.00 8.62 ± 0.00 24.44 ± 0.00 31.44 ± 0.00 70.91 ± 0.00 5.29 ± 0.01 15.18 ± 0.00 33.4 ± 0.00

Plectonema nostocorum Bornet ex Gomont 2.96 ± 0.00 10.01 ± 0.02 8.03 ± 0.00 3.96 ± 0.00 34.94 ± 0.00 114.60 ± 0.01 5.24 ± 0.00 17.76 ± 0.02 39.18 ± 0.02

Phormidium bohneri Schmidle 0.42 ± 0.00 28.14 ± 0.00 6.66 ± 0.00 16.47 ± 0.00 206.14 ± 0.00 91.87 ± 0.00 1.91 ± 0.00 18.49 ± 0.01 21.56 ± 0.00

Lyngbya truncicola Ghose 7.44 ± 0.00 26.44 ± 0.00 14.19 ± 0.00 8.89 ± 0.05 115.22 ± 0.01 151.00 ± 0.01 11.52 ± 0.04 15.23 ± 0.00 53.20 ± 0.01

Phormidium fragile (Meneghini) Gomont 0.29 ± 0.00 6.63 ± 0.00 4.12 ± 0.01 2.04 ± 0.00 33.48 ± 0.00 47.06 ± 0.01 7.00 ± 0.00 76.24 ± 0.00 19.13 ± 0.02

Microchaete uberrima Carter, N. 0.45 ± 0.00 52.62 ± 0.00 79.55 ± 0.03 0.48 ± 0.00 102.99 ± 0.00 68.55 ± 0.03 2.76 ± 0.00 22.85 ± 0.00 45.33 ± 0.04




63

Figure 3.A, B = Growth behaviour and microphotograph of Anabaena naviculoidesC, D = Growth behaviour and microphotograph of Anabaena variabilisE, F = Growth behaviour and microphotograph of Anabaena fuelleborniiG, H = Growth behaviour and microphotograph of Nostoc spongiaeformeI, J = Growth behaviour and microphotograph of Phormidium tenueK, L = Growth behaviour and microphotograph of Plectonema nostocorumM, N = Growth behaviour and microphotograph of Phormidium bohneriO, P = Growth behaviour and microphotograph of Lyngbya truncicolaQ, R = Growth behaviour and microphotograph of Phormidium fragileS, T = Growth behaviour and microphotograph of Microchaete uberrima



64

the dietary carotenoid provided and thus carotenoids are frequently used in dietary additives for poultry and aquaculture farming (Hirschberg & Chamoritz 1994). The spectrum of carotenoids in Anabaena variabilis and three species of Phormidium showed β- carotene as the major pigment (Healey 1968).

Similarly, carotenoid composition of Anabaena flos-aquae and three other species of Phormidium also showed the presence of β-carotene as the major carotenoid in all species (Hertzberg et al. 1971). Cyanobacteria are considered a system of photoproduction of ammonia by their diazotrophic potential. A semi-continuous ammonia production system employing the heterocystous cyanobacterium Cyanospira rippkae was tried (Vincenzini et al. 1986) using L-Methionine DL-sulfoxamine (MSX), an inhibitor of the ammonia assimilating enzyme, glutamine synthetase. Similarly, mutant strain of Anabaena variabilis fixed N2 and liberated NH4

+ into the media (Kerby et al. 1986). Cyanobacteria are usually non-pathogenic and have high nutritive value, rich in carbohydrates, proteins, lipids, minerals and vitamins (Cannell 1989).

Genetic approaches of potential isolates to construct cyanobacterial isolates could be used to improve to natural colouring product from several living micro-organisms. These ten (10) isolates were found at par with already commercialized particularly for natural colouring materials for industrial applications (‘Linablue’, a product from Dainippan Ink and Chemicals Inc, Japan). Since, these isolates were found to contain more of pigments, total soluble proteins, ammonia and carotenoids, they are considered as potent candidates for alternative resources of fulfilling gap of malnutrition, biofertilizers, and colouring agents (Table 2, 3, & 4). Therefore, it is necessary that a detailed survey of the surrounding habitats are made to identify the available cyanobacterial species and subsequently to isolate, purify and establish a culture collection which could be used for further studies.

CONCLUSIONThe present study focused mainly on the cultural studies and biochemical characterization of the potent isolates from North-East region of India. Only scattered information were available on the diversity and cultural behaviour of cyanobacteria from the North-Eastern region of India (Tiwari & Singh 2005; Deepa et al. 2010). Molecular typing and distribution of filamentous heterocystous cyanobacteria isolated from two distinctly located regions in North-Eastern India has been carried out (Chingkheihunba & Arvind 2011). The present findings indicates that producing mass culture of cyanobacteria

for industrial purposes represents a novel biotechnology which naturally prompts questions concerning the future for such kinds of the endeavour. Therefore, it is thus imperative that the cyanobacteria possess immense biotechnological potentials for applications towards human welfare. However, except for the use of Spirulina as a health food, there are no other major product(s) from cyanobacteria is popularized in worldwide. This is due to the limitations in the economic production of cyanobacterial products in the industrial scale.

Basically, many of the metabolites are produced by the organism(s) in low amounts, there is no mass cultivation technology evolved for such potential cyanobacteria and in many cases, the method of industrial extraction is not optimized. Intensive research is thus warranted to understand many of the basic aspects pertaining to the production of a metabolite with the concurrent evolution of applied research towards the large production of the products. Also, further studies are needed for the need of potent cyanobacteria from this region over the long term and the use of products of biological, non-toxic products from these isolates which is still a long way down the road.

ACKNOWLEDGEMENT We are thankful to Dr. N. C. Talukdar, Director, IBSD, Imphal, Manipur, India for Laboratory assistance, Indian Council of Agricultural Research (ICAR) and Department of Biotechnology (DBT) Government of India for providing financial assistance. Ojit Singh K & Gunapati Oinam are also thankful to lab mates especially to Laxmipriya, Avijeet, and Indrama for their tireless support.

REFERENCESA N A G N O S T I D I S K , K O M A R E K J . 2 0 0 5 .

Cyanoprokaryota 2. 2nd part: Oscillatoriales 2. 1st ed. Spektrum Akademischer Verlag, Italy. p. 8-660

BAUERNFEIND JC. 1981. Carotenoids as colourants and Vitamin A Precursors: technologica and nutritional applications. Academic Press. Inc. New York.

BENNETT A, BOGORAD L. 1973. Complementary chromatic adaptation in a filamentous blue-green alga. J Cell Biol 58: 419-435.

B O R O W I T Z K A M A . 1 9 8 8 . Vi t a m i n s a n d f i n e c h e m i c a l s f r o m m i c r o a l g a e . I n : Microalgal Biotechnology (eds). Borowitzka MA, Borowitzka LJ. Cambridge University Press. p.



65

153–196.

BOROWITZKA MA. 1994. In: proceedings of the First Asia-Pacific Conference on Algal Biotechnology (Eds.) Phang SM, Lee YK, Borowitzka MA, Whitton BA. University of Malaya, Kuala Lumpur. p. 5-15.

CANNELL RJP 1989. Algal biotechnology. In: Weetall H. (Ed.) Applied Biochemistry and Biotechnology. Totowa, NJ, US: The Humana Press Inc. p. 85-105

CARMICHAEL WW. 2001. Health effects of toxin-producing cyanobacteria: “The cyanoHABs” Human and Ecological Risk Assessment 7(5): An International Journal. p. 1393-1407.

CHACON-DE-POPOICI G. 1994. Plankton blooms, their effects on the marine organisms for the quantitative analysis of microcystins in cyanobacterial cells. Phycologia 35(6): 57-61.

CHINGKHEIHUNBA A, ARVIND KS. 2011. Molecular typing and distribution of filamentous heterocystous cyanobacteria isolated from two distinctly located regions in North-Eastern India. World J Microbiol Biotechnol. Springer Publications Online version. DOI 10.1007/s11274-011-0684-8

CODD GA. 1997. Cyanobacterial blooms and toxins in fresh, brackish and marine waters. Harmful Algae. Proceedings of the VIII International Conference on Harmful Algae. (Eds.) Reguera B, Blanco J, Fernandez ML & Wyatt T. p. 13-17.

COHEN Z. 1986. Products from Microalgae. In: Handbook of Microalgal Mass Culture. (Ed.) Richmond A. Boca Raton Florida, USA: CRC Press. p. 421-454.

DAINIPPON INK, CHEMICALS INC. 1980. Production of highly purified alcoholophilic phycocyanin. Japanese Patent 80, 77, 890.

DAINIPPON INK AND CHEMICALS INC.1981. Cosmetics containing water soluble phycocyanin. Japanese Patent 79-138755.

DEEPA DEVI S, THINGUJAM INDRAMA & TIWARI ON. 2010. Biodiversity Analysis and Reproductive/Cultural Behaviour of Cyanobacteria of North-East Region of India having Acidic Properties. The International Journal of Plant Reproductive Biology 2(2): 127-135

DESIKACHARY TV. 1959. Cyanophyta. Indian Council of Agricultural Research, New Delhi, India. p. 3-616.

DE VRIES SE, GALEY FD, NAMIKOSHI M, WOO JC. 1993. Clinical and pathological findings of blue green algae (Microcystis aeruginosa) in toxication in a dog. J. Vet. Diagn. Investig. 5(3): 403-408.

EMODI A. 1978. Carotenoids: Properties and Applications.

Food Technol 32: 38-42.

GUERRERO MG, RAMOS JL, LOSADA M. 1982. Photosynthetic production of ammonia. Experientia 38. p.53.

GUSTAFSON KR, CARDELLINA JH, FULLER RW, WEISLON OS, KISER RF, SNADER KM. 1989. Antiviral sulfolipids from cyanobacteria (blue-green algae). J Nat Caner Inst 81(16): 1254-1258.

HALL DO, GRASSI G. 1989. Photoconvertion Processes for Energy and Chemicals (eds. Hall DO, Grassi G). Elsevier. Amsterdam. p. 28-45.

HEALEY FP. 1968. The carotenoids of four Blue-green algae. J Phycol 4(2): 126-129.

HERBERT D, PHIPPS PJ, STRANGE RE. 1971. Chemical analysis of microbial cells. In: Methods in microbiology Vol. (v) B (ed.) Morris JR, Ribbons DW. New York: Academic Press. p. 209-234.

HERTZBERG S, LIAAEN-JENSEN S, SIEGELMAN HW. 1971. The carotenoids of blue-green algae. Phytochemistry 10(12): 3121-3127.

HIRSCHBERG J, CHAMORITZ D. 1994. In: The Molecular Biology of Cyanobacteria. Bryant DA. Dordrecht (ed). The Netherlands: Kluwer Academic Publishers. p. 559-579.

JENSEN A. 1978. Chlorophylls and carotenoids. In: Hellebust JA, Craige IS. (ed). Hand book of Phycological Methods. Physiological and Biochemical Methods. Cambridge: Cambridge University Press. p. 59-70.

KERBY NW, MUSGRAVE SC, ROWELL P, S H E S TA K O V S , S R E WA R D W D P. 1 9 8 6 . Photoproduction of ammonium by immobilized mutant strains of Anabaena variabilis. Appl Microbiol Biotechnol 24(1): 42-46.

MCKINNEY G. 1941. Absorption of light by chlorophyll solutions. J Biol Chem 221: 315-322.

MITSUI A, MURRAY R, ENTENMANN B, MIYAZAWA K, POLK E. 1981. Utilization of marine blue–green algae and macroalgae in warm water mariculture. In Biosaline Research. A Look to the Future (ed. San Pietro A.). New York: Plenum Press p. 215–225.

MIURA Y, SODE K, NARASAKI Y, MATSUNGAGA T. 1993. Light induced antimicrobial activity of extracts from marine Chlorella. J Mar Biotechnol 1(3): 143-146.

MOORE RE. 1981. Constituents of blue-green algae. In: Marine Natural Products: Chemical and Biological Perspectives 4: 1-52 (Ed.): Scheuer PJ. New York: Academic Press. p. 1-52.



66

PRABHAHARAN D, SUBRAMANIAN G. 1995. Hydrogen photoproduction by marine cyanobacteria Dicothrix bauriana BDU 40481. Physiol Mol Biol Plants. 1: 45–57.

RAMOS JL, GUERRERO MG, LOSADA M. 1987. Factors affecting the photoproduction of ammonia from dinitrogen and water by the cyanobacterium Anabaena sp. Strain ATCC 33047. Biotechnol Bioeng 29(5): 566-571.

SEKAR S, SUBRAMANIAN G. 1998. A method of mass cultivation of the marine cyanobacterium Phormidium valderianum BDU 30501 for the production of blue natural colourant phycocyanin. In : Cyanobac te r i a l b io techno logy. ( eds ) . Subramanian G, Kaushik BD, Venkataraman GS. New Hampshire USA: Science Publishers. p. 305-309.

SKULBERG OM. 2000. Microalgae as a source of bioactive molecules experience from cyanophyte research. J App Phycol 12: 341-348.

SOLARZANO L. 1969. Determination of ammonia in natural waters by the phenol hypochlorite method. Limnol Oceanogr 4: 799-801.

SONG T, MARTENSSON L, ERIKSSON T, ZHENG W, RASMUSSEN U. 2005. Biodiversity and seasona l va r i a t ion o f the cyanobac te r i a l assemblage in a rice paddy field in Fujian, China. The Federation of European Materials Societies Microbiology Ecology 54: 131-140.

SPIRO RG. 1966. Analysis of sugars found in glycoproteins. Methods in Enzymology 8: 3-26.

STALEY JT, STANLEY PM. 1986. Potential commercial applications in aquatic microbiology. Microb Ecol 12: 79-100.

SUBRAMANIAN G, UMA L. 1996. Cyanobacteria in pollution control. J Sci Ind Res 55: 685–692.

SUNDARARAMAN M, SUBRAMANIAN G, AVERAL HI, AKBHARSHA MA. 1996. Evaluation of the bioactivity of marine cyanobacteria on some biochemical parameters of rat serum. Phytotherapy Res 10: 9-12.

TIWARI ON, TOMBI SINGH H. 2005. Biodiversity of cyanobacteria in Loktak lake and rice fields of Manipur, having acidic properties. Proc Nat Acad Sci India 75(B) III: 209-213.

VINCENZINI M, DE PHILIPPIS R, ENA A, FLOREZANO G. 1986. Experimentia 42: 1040-1043.

WALSH K, JONES GJ, DUNSTAN RH. 1997. Effects of irradiance on fatty acid, carotenoid, total protein composition and growth of Microcystis aeruginosa. Phytochemistry (UK) 44: 817-824.

WIKSTROM P, SZWAJEER E, BRODELIUS P , N I L S S O N K N , M O S B A C H K . 1997. Formation of alpha-keto acids from amino acids using immobilized bacteria and algae Biotech Lett p. 4-153.

new record of potential cyanobacteria from indian region

Documents