Isolation and optimization of cultural conditions for exopolysaccharide production from Blue Green Algae.
- Mohanapriya R and Geetharamani D
Polysaccharides are renewable resources representing an important class of polymeric materials of biotechnological interest. They offer a wide variety of potentially useful products to mankind. Extracellular polysaccharides of microbial origin are of interest due to their novel functionality, reproducible physio-chemical properties, stable cost and supply. Cyanobacteria exhibit high growth rate and are more willing to manipulate conditions for enhancing growth or Exopolysaccharide (EPS) production. When compared to macro algae and higher plants microbial exopolysaccharide has received increasing attention recently due to their potential application as industrial gums, bio flocculants, soil conditioners and bio absorbents. In the present study, blue green algae isolated from Noyyal river in Coimbatore, Tamil Nadu were screened for EPS production by Negative staining. Among the ten isolates screened one isolate identified as Chroococcus sp was found to produce polysaccharide. Optimization of cultural conditions for the production of exopolysaccharide by Chroococcus sp was carried out. The isolate was subjected to mass cultivation in the optimized medium. The exopolysaccharide was extracted. The total carbohydrate and total protein were determined by phenol sulphuric acid method and Lowry et al., method. A yield of 55mg/l of exopolysaccharide was obtained.
Key words: Blue-green algae, Chroococcus sp, Exopolysaccharide (EPS).
Cyanobacteria or Blue-green algae are photosynthetic prokaryotic organisms which are either unicellular or filamentous structures. These organisms are found in both freshwater and marine water environment. Some Cyanobacteria grow in association with certain green algae, liverworts, water ferns, and Angiosperms (Bold and Wynne,1985).
Cyanobacteria have been used in many applications and for a long time they have been known to produce large exopolysaccharide (Drews and Weckesser, 1982). Microbial exopolysaccharide are a class of polymeric materials with potent biotechnological application and are renewable materials (De Philippis et al.,2001). These has been a growing interest in the production of exopolysaccharide their varied applications. They are mainly produced by bacteria and Cyanobacteria (Misra and Jha, 2009). The exopolysaccharide find application like industrial gums, soil conditioners, bioflocculant, biosorbants and to prevent adherence of pathogenic organisms. (Bertocchi et al., 1990; Morvan et al.,1997; De Philippis and Vincenzini, 1998). Okutani, 1984 reported that polysaccharides produced by marine Vibrio sp and Pseudomonas sp exhibited antiviral, antitumor and immunostimulant activities. Colliec Jouault et al., 2001 stated that Alteromonas infernus produce heparin like exopolysaccharide with anticoagulant property.
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Many cyanobacteria have mucilaginous external layers such as capsule, mucilage, sheath, glycocalyx or slime. These external layers are polysaccharide in nature. There are three types of polysaccharide. A sheath is a thin uniform structured external layer immediately next to the outer membrane that contains either concentric or radial fibres, according to the strains. A capsule or slime polysacchradie layers (CPS) are the more outer unstructured zones and certain soluble polysacchraides (released polysacchraides, RPS) are released by many cyanobacteria into media (Bold and Wynne, 1985; Bertocchi et al., 1990).
In the present study cyanobacterial strains isolated from Noyyal river was screened for exopolysaccharide production. Optimization of cultural conditions was determined and mass cultivation was done using the optimized medium. The exopolysaccharide was extracted. Total protein and carbohydrate content were determined.
2.MATERIALS AND METHODS
2.1 Collection of samples
Samples were collected from four different locations of Noyyal River. Algal samples were collected using sterile 50 ml plastic bottles. All samples for chemical analysis were either analyzed immediately or stored at -20°C.
2.2 Isolation of microalgae
The collected samples were initially inoculated into five different 100ml conical flask containing 50ml of BG 11 medium. After inoculation the flasks were incubated at 24+/-2°C under 37.5 µmol-1m-1sec-1intensity with 16:8h photoperiod for 15 days (Dayananda et al., 2010).
2.3 Purification of Microalgal strains
Microalgae was isolated and maintained in axenic cultures which are based on serial dilution culture technique and agar plate method as described by Gopinathan (1996). The mixed microalgal cultures were then serially diluted using respective culture medium in which growth was observed. After serial dilution, the flasks were incubated at the above said condition for the purification of microalgae. From the serially diluted flask, the samples were inoculated onto agar plates with 10mg/l of cyclohexamide for final purification process. The pure cultures thus obtained were sub-cultured and maintained.
2.4 Screening of isolated strain for exopolysaccharide production by Negative staining
Negative staining was performed by mixing a 50 µl of algae cultures with India ink dye. After mixing a smear was made with the preparation, air dried and observed under the microscope (45 X objective lens) (Plude et al., 1991). The isolate which showed visible halo around the colonies were subjected to further study.
2.5 Optimization of growth medium for Exopolysaccharide production
Growth medium was optimized for temperature, pH, carbon source and time of incubation (Sunil T pawar et al .,2013).
2.6 Mass cultivation for Exopolysaccharide production
The isolate was subjected to grow in the optimized medium. The maximum yield of exopolysaccharide at time intervals was determined.
2.7 Extraction of exopolysaccharide
The exopolysaccharide was extracted by adding 12 ml of ice cold ethanol to the medium containing 100ml of grown cells and incubated for 4°C overnight. The exopolysaccharide was precipitated and collected at 10000 rpm for 20 minutes. The pellet thus obtained was dried at 100°C and weighed (Sunil T Pawar et al., 2013). The extraction was done at regular periodic intervals and maximum amount of polysaccharide produced at particular time was determined.
2.8 Estimation of carbohydrate and protein content in extracted exopolysaccharide
The total carbohydrate content of the extracted polysaccharide was estimated using phenol sulphuric acid method. The protein content of extracted polysaccharide was estimated using Lowry et al ., method.
3. RESULT AND DISCUSSION
A wide range of Cyanobacteria are present in water samples. From twenty water samples, ten Cyanobacteria were isolated in pure form by serial dilution techniques. All isolates were checked for exopolysaccharide production by Negative staining using India ink. It was found that isolate c produced a clear halo around the colonies which indicates the production of exogenous slime layers (Fig 1). By microscopic observation of morphology, the isolate was found to be Chrococcus sp based on David et al., 2011.
Fig 1: a) Microscopic observation of isolate c and b) negative staining of isolate
pH, temperature, carbon source and time of incubation for the exopolysaccharide production by the isolate was optimized. It was found that the exopolysaccharide production was maximum at a pH of 7.5, temperature 25°C, with 1% sucrose and an incubation time of 9 days (Fig 2).
Mass cultivation of exopolysaccharide was done using optimized medium and exopolysaccharide was extracted at regular time intervals. The maximum amount of polysaccharide produced was found to be 55mg/l at 9th day of incubation (Fig 3). The carbohydrate content found in exopolysaccharide was found to be 64 % and the protein content was 11.8%.
Fig 2: Optimization of growth parameters- a) temperature b) pH c) Carbon source d) salt concentration
Figure 3: Amount of Exopolysaccharide produced at different days of incubation in the optimized medium.
Ten different cyanobacteria strains were screened for exopolysaccharide production. Among ten isolates isolate c found to produce maximum amount of exopolysaccharide at 9th day of incubation and isolate was identified as Chroococcus sp based on their morphology. Optimization was done to increase the production of exopolysaccharide using different parameters like pH, temperature, carbon sources and time of incubation. It was found that Chroococcus sp able to produce high amount of exopolysaccharide (55mg/ml) in a growth medium containing 1% sucrose, at a pH of 7.5 and a incubation temperature of 25°C in 9 days.
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