The habitats ranging from oceans to the fresh waters constitute many life forms that include one oldest life form called as cyanobacteria or blue green algae. Cyanobacteria is a gram-negative bacteria and they are also called as photosynthetic autotrophs. Cyanobacteria are capable of fixing nitrogen from the atmosphere and hence they are considered as beneficial in agriculture as bio-fertilizers. Blue green algae are known to be producing pharmaceutical products like hormones, antibiotics, iron chelators and toxins.
Nostoc ellipsosporum is a filamentous form of cyanobacteria that is reported to be able to produce medicinal products beneficial for humans. Hence, they are considered important in biotechnology. The protein molecule called as cyanovirin that is produced from N.ellipsosporum is found to be helpful in treating H1N1 and HIV diseases.
Heterocysts are cells present in cyanobacteria to fix atmospheric nitrogen. The nitrogen fixed by heterocysts will be transferred to the vegetative cells as amino acids for metabolic purpose when there is no nitrogen in the growth medium. Various studies have reported that growth, metabolism and photosynthesis of the cyanobacteria are affected by light, temperature, pH and nutrients in the medium. So, to increase the cell mass the nutrient medium can be improved.
Phaseolus vulgaris consists of phytohaemagglutinin or PHA. PHA is a lectin that has its role in cell proliferation and defence mechanisms. The present study concentrates on protein synthesis and growth of Nostoc ellipsosporum NCIM 2786 on the Fog’s medium which is supplemented with the extract of Phaseolus vulgaris rich in phytohaemagglutinin and glucose. Statistical analysis of medium components was performed by Plackett-Burman method.
Microorganism: The Nostoc ellipsosporum NCIM 2786 strains are gathered from National chemical laboratory and national collection of industrial microorganisms at Pune in India. The culture was obtained in Fog’s medium.
Growth medium: Fog (G-) medium comprised of calcium, magnesium and potassium salts with micronutrients and EDTA. Fog (G+) medium has glucose added to all other components of Fog (G-) composition.
Crude extract of Phaseolus vulgaris: The seeds of this plant were weighed 10 grams and they are homogenized in 100 ml of distilled water. The paste is again dissolved in 100 ml of DW and mixed well. This extract is centrifuged and the supernatant is collected and used as P.vulgaris extract.
Analytical method: Nostoc ellipsosporum was grown in Fog’s G- medium and Fog’s G+ medium supplemented with light. The growth of the Nostoc cells was observed by measuring the absorbance using spectrophotometer at 600 nm wavelength. Lowry method was used for estimation of protein content. The variables present in the growth medium influencing the growth of the bacteria and their significance are estimated using Placket-Burman method after several repetitions of the experiment. The significance of variables in the experiment is determined by the equation Y = β0 + ∑ βi Xi .
Here, Y is the estimated response, βi is the regression coefficient and Xi is the independent variable.
Results of the study
Influence of glucose on the growth of N. ellipsosporum
The growth of Nostoc ellipsosporum was observed in both Fog’s G- and G+ media for 10 to 12 days. The growth was found maximum after 10 days. The culture turned green conspicuously after 8 days in the medium supplemented with glucose. The non-glucose medium showed slight green colored growth of bacteria compared to the colorless control medium without any bacterial inoculation. The Nostoc cells were collected and investigated under scanning electron microscope.
The heterocysts were clearly seen in the filaments that were grown in glucose+ medium while the heterocysts were not clearly formed in the filamentous bacterium grown in glucose- medium. The absorbance values were similar for bacteria grown in both the media after 9 days although the heterocysts were well developed in G+ medium. Though these cyanobacteria are phototrophs, their cells grow profusely and synthesize proteins inside the cells when they grow in medium with nutrients and glucose. The heterocyst formation with proper morphology was observed to be vital for the protein synthesis by Nostoc ellipsosporum as the proteins are essential for the generation of pharmaceutical bio-products. The glucose in the medium will be used for the synthesis of polysaccharide envelope for the heterocysts.
Impact of PHA on growth of Nostoc cells
The addition of 1 percent of P.vulgaris extract to 100ml of Fog’s medium showed improved cell growth. The Fog’s medium without the Phaseolus extract was also inoculated with bacteria where the cell growth was not observed properly. Both of the media were devoid of glucose. The medium with extract addition could show change into green color while the one without the extract did not show change in color. The cell proliferation and protein synthesis might be triggered by the stimulation of cell surface receptors by PHA.
Screening of medium variables
Plackett-Burman design was used to screen the significant medium variables. Out of the 12 experiments, responses extending between 0.238mg/ml and .672mg/ml were observed. Regression coefficients and main effect values were calculated. The results showed that P.vulgaris extract and glucose could show positive impact on protein synthesis in Nostoc cells. Other medium variables showed negative impact on protein synthesis. All the medium main effect variables were significant. ANOVA was performed with correlation coefficient of the main effect values as 0.941 and the model was found to be fit by 94.1 percent.
Srivastava Anshuman, Mittal Deepika, Govindasamy Sharmila, Chandrasekharan Muthukumaran. Effect of glucose and phytohaemagglutinin (PHA) rich Phaseolus vulgaris extract on growth and protein synthesis of pharmaceutically important cyanobacteria Nostoc ellipsosporum NCIM 2786. Journal of Genetic Engineering and Biotechnology (2013) 11, 33-37.
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