Enhancing pigment production by a chromogenic bacterium (Exiguobacterium aurantiacum) using tomato waste extract: A statistical approach

Abstract

There is a high demand for microbial pigments as a promising alternative for synthetic pigments, primarily for safety and economic reasons. This study aimed at the optimization of yellowish-orange pigment production by Exiguobacterium aurantiacum using agro-waste extracts as a growth substrate. Air samples were collected using the depositional method. Pure cultures of pigment producing bacteria were isolated by subsequent culturing on fresh nutrient agar medium. The potent isolate was identified using MALDI-TOF technique. Screening of culture conditions was done via Plackett-Burman design that highlighted culture agitation rate, initial medium pH, and yeast extract concentration as the most significant variables (p < 0.0001) in influencing pigment production with further optimization step using response surface methodology. Among the tested agro-waste decoctions, tomato waste extract was selected for fermentation due to higher optical density of the isolate when cultivated in it compared to the other agro-waste extracts. Under optimized conditions, 0.96 g/L of pigment was extracted from 4.73 g/L of culture biomass, representing a 1.6-fold increase compared to un-optimized conditions. Spectroscopic and chromatographic analyses confirmed the presence of various functional groups, with carotenoids identified as the primary compounds responsible for the yellowish-orange pigmentation. These findings demonstrate the feasibility of enhancing bacterial pigment production using agro-waste substrates, highlighting its potential for large-scale industrial applications.

Fig 1. Isolation of pure colony from mixture of colonies grown on nutrient agar medium using streaking method.

Fig 2. MALDI-TOF-MS spectrum of yellowish-orange pigment producing bacteria, identified as Exiguobacterium aurantiacumshowing distinct mass-to-charge ratio (m/z) peaks associated with bacterial proteins.

Fig 3. Half-normal plot for the standardized effects of process variables on OD_{600 nm} value, highlighting critical factors for optimization.

Fig 4. Pareto chart depicting the influence of process variables on OD₆₀₀ value in descending order, bars representing each variable’s contribution to the overall effect.

Fig 5. Response surface optimization graphs, panels (A)-(F).

Panel (A) shows the relationship between agitation speed (rpm) and pH on OD value. Panel B illustrates effects of yeast extract concentration and initial medium pH on OD value. Panels C and D elucidate the combined effects of agitation versus pH and yeast extract versus pH on culture biomass yield, and panels E and F demonstrates how pigment yield is influenced by agitation rate, pH, and yeast extract concentration.

Fig 6. Smooth line graph showing OD₆₀₀ variation over time of Exiguobacterium aurantiacum at optimized process conditions using TWE as cultivation substrate with standard deviation represented as error bars around the line.

Fig 7. IR spectrum of pigmented compound extracted from Exiguobacterium aurantiacum, depicting characteristic absorption peaks corresponding to various functional groups present in the compound.

Fig 8. Absorption spectrum of pigment extracted from Exiguobacterium aurantiacum scanned from 350-750 nm using UV-visible spectrophotometer.

Fig 9. LC-MS analysis of Carotenoid compounds extracted from Exiguobacterium aurantiacum.

Panel A illustrates the ion intensity detected over time, panels B and C reveal peaks corresponding to the mass-to-charge ratios (m/z) of the detected carotenoid molecules.