Peanut seed, mustard seed, and sesame seed oil cakes were purchased from a local oil factory located in Mumbai. All the chemical and reagents used were of AR grade and purchased from Himedia, Mumbai. S. marcescens ATCC 13880 was a gift sample from K. J. Somaiya Microbiology Department, Mumbai. The culture was maintained on Luria Bertanni (LB) agar medium and sub-cultured after every 15 days. The slants were stored at 4 °C in a refrigerator.
Prodigiosin production and its estimation by UV-Vis spectroscopy
In order to compare pigment production, both liquid and solid forms of media were screened for pigment production. Briefly, 20 ml of nutrient broth (NB) was inoculated with fresh 5% culture. Similarly, nutrient agar (NA) plate was spread with 5% of culture. Both the media were incubated for 72 h and evaluated for pigment production. The quantitative determination of the red pigment was done by measuring the absorbance at 530 nm using double beam UV-Visible spectrophotometer as suggested by Chen et al. [17]. In case of solid medium, the culture was scraped and suspended in 20 ml distilled water (D/W). For the extraction process, 5 ml from above broth was taken in a test tube, and 4 ml of methanol was added. The mixture was vigorously vortexed for 2 min. The solution was then centrifuged for 10 min at 6000 rpm. Following the above step, 0.8 ml supernatant was further mixed with 0.2 ml of 0.05 N HCl: methanol mixture (4:1 v/v). The absorbance of the resulting solution was then measured at 530 nm [17].
Optimization of culture growth conditions for prodigiosin production
Sterile nutrient agar was used as the basal medium, as the culture failed to grow in broth. Important parameters, like pH (6.5, 7, 7.5, and 8), temperature (25, 30, and 37 °C), and incubation time (24, 48, 72 h) were checked for optimum pigment production.
The effect of carbon source (each 2% glucose, sucrose, lactose, and fructose), nitrogen source (1% of peptone, yeast extract, beef extract, casein hydrochloride) were also studied, respectively. After optimizing each parameter for maximum prodigiosin production, the same was selected and maintained throughout the study.
For optimization of fatty acid sources, natural substrates, like oil seed cakes of peanut, mustard, and sesame, were crushed in a mixer and sieved to fine particles. Fine particles of oil seed cakes were stored at 4 °C until further use. Nutrient agar supplemented with 2% oil seed cake powder was used for prodigiosin production using S. marcescens strain. The pH was adjusted to 7.5 using 1 N NaOH. All the media were autoclaved at 121 °C for 20 min. All experiments were carried out in triplicates. The unconventional sources which gave higher production of prodigiosin such as peanut and sesame seed powder were further screened for their concentration in the range from 1 to 4%.
Effect of solvents in prodigiosin extraction
Solvents, like ethanol, methanol, acetone, and dimethyl sulfoxide (DMSO), were used to extract crude prodigiosin. Briefly, the culture was scraped from Petri plates and washed with 20 ml of D/W. The washed pellet was dissolved in 4 ml each of the mentioned solvents and absorbance reading was noted at 530 nm. Higher absorbance reading exhibited higher pigment extraction.
Scale-up studies for prodigiosin biosynthesis and biomass estimation
After optimization of all the parameters, prodigiosin biosynthesis was determined by upscaling the protocol using 1 L media. The medium was prepared using optimized parameters, like 4% peanut oil seed cake powder, 2% sucrose, 1% casein hydrolysate, pH 7.5, temperature 28 °C, and incubated for 72 h. Briefly, the culture was scraped off from Petri plates and suspended in 20 ml of D/W. The cells were centrifuged at 10 min at 6000 rpm and suspended in D/W, vortexed, and centrifuged again to obtain pellet. The pellet was dissolved in 4 ml of methanol, vortexed, and centrifuged again. The resulting pigmented supernatant was collected by filtering through 0.2 μ Whattman filter paper. The filtrate obtained was dried in a hot air oven at 55 °C overnight. The crude powder was again suspended in 4 ml of chloroform and dried again. The obtained powder was then weighed and expressed as gram per liter.
Purification, confirmation, and characterization of pigment
In this study, the pigment pellets were collected from 1 L media by scrapping the Petri plates. The washed pellets were then dissolved in 50 ml of acidified methanol (4 ml + 96 ml methanol) followed by ultrasonication for 30 min. The supernatant was left to stand for 1 h. The supernatant was then collected by rotary evaporation to obtain the crude form of the pigment. The crude form of pigment was then purified using a silica gel column. The pigment was eluted using petroleum ether for five volumes followed by petroleum ether: ethyl acetate (1:1 v/v). The red elute was collected and concentrated using rotary evaporator at 40 °C [19].
The pigment was confirmed by UV wavelength scan, thin layer chromatography, and Gas chromatography-mass spectrophotometry. Two purified pigment extracts (3 ml) were separately dissolved in methanol and dimethyl sulphoxide (DMSO) solvents and scanned under UV-spectrophotometer (Beckman DU-800) in the range 200–800 nm for maximum absorbance.
The purified pigment extracts dissolved in methanol and DMSO separately were then characterized using subjected thin layer chromatography using a developing solvent (70% acetone + 30% hexane). The Rf values were compared to values reported in the literature. The pigment extract was dissolved in 5 ml methanol and subjected to mass spectral analysis (GC-MS) to determine the molecular mass of the pigment by comparing prodigiosin derivatives as reported in the literature. Vitamin A, a chemical analog of prodigiosin with similar pyrrole ring structure, molecular weight (333 amu), was used as a reference compound. The mass match was carried out by a computerized search with already registered prodigiosin-like compounds [20].