Bioprocess for the simultaneous production of polyhydroxybutyrate and violacein pigment from himalayan bacterium iodobacter sp. PCH 194
The present disclosure provides a bioprocess for the simultaneous production of Polyhydroxybutyrate (PHB) and Violacein pigment in a single fermentation using a Himalayan bacterial isolate Iodobacter sp. PCH 194, having accession number MTCC 25171. PHB has plastic properties, renewable origin and bio-degradable nature. It can be used for various packaging applications replacing the petrochemicals-based plastics, thus providing greener alternative to environment. Violacein pigment has anti-oxidant, anti-tumoral, anti-bacterial, and photo-protective properties and can be used in cosmetics and pharmaceuticals applications. The present disclosure provides production process of two valuable products i.e. PHB and violacein pigment in a single bioprocess, therefore, is economically attractive, time saving, and commercially feasible process.
1 . A bioprocess for the simultaneous production of polyhydroxybutyrate (PHB) and violet-blue violacein pigment using the isolated bacterial strain of Iodobacter sp. MTCC 25171, the process comprising the steps:
i) inoculating a loopful of Iodobacter sp. MTCC 25171 in 50 mL sterile nutrient broth medium and incubating at a temperature of 20±2° C. for 32 to 36 hours, at 100 to 200 rpm to obtain a seed culture for inoculating the production medium;
ii) inoculating 3 to 5% v/v of the seed culture as obtained in step [i] to a sterile production medium and incubating for 36 to 100 hours at 100 to 200 rpm at a temperature of 20±2° C. to obtain a biomass;
iii) harvesting the biomass as obtained in step [ii] to obtain a pigmented cell pellet by centrifugation or microfiltration;
iv) suspending the pigmented cell pellet as obtained in step [iii] in methanol or ethanol, in a volume ratio of 1:3 to 1:5 and mixing well for 10 to 15 minutes at room temperature followed by centrifugation to obtain a pigment in the supernatant; and repeating the step until no visible violet colour remains in the cell pellet to obtain a colourless cell pellet;
v) concentrating the supernatant as obtained in step [iv] using vacuum evaporation to obtain the desired violacein pigment;
vi) suspending the colourless cell pellet as obtained in step [iv] in 3 to 5 times the volume of 1:1 trichloromethane and bleaching solution, followed by incubating overnight at temperature ranging from 30 to 40° C. under continuous shaking to obtain a mixture;
vii) allowing the mixture as obtained in step [vi] to stand for 10 to 15 minutes and pipetting out the lower organic layer in a test-tube and then adding 3 to 5 times the volume of chilled methanol, followed by mixing it well and keeping at temperature ranging from minus 4 to minus 20° C. for 30 to 60 minutes for precipitation to obtain a reaction mixture;
viii) centrifuging the reaction mixture as obtained in step [vii] to collect the pellet as precipitate and removing the residual solvent;
ix) drying the pellet as obtained in step [viii] and re-dissolving it in trichloromethane; and
x) repeating steps vi to viii and finally collecting the pellet of Polyhydroxybutyrate by allowing the residual solvent to evaporate.
2 . The process as claimed in claim 1 , wherein the 36 to 100 hour incubation of step ii) is performed at 20° C. and 150 rpm.
3 . The process as claimed in claim 1 , wherein the yield of PHB and violacein pigment in 1 liter culture after 96 hours of incubation is in range of 3 to 5 g/L, and 0.5 to 0.8 g/L, respectively.
4 . The process as claimed in claim 1 , wherein the 36 to 100 hour incubation of step ii) is carried out in a fermenter while maintaining 20% dissolved oxygen and 150 rpm.
5 . The process as claimed in claim 1 , wherein the yield of PHB and violacein pigment in 7.5 or 10 liter working volume of fermenter after 96 hours of incubation is in range of 7 to 10 g/L, and 0.8 to 1.5 g/L, respectively.
6 . The process as claimed in claim 1 , wherein the PHB obtained is useful for the preparation of biodegradable bio plastics.