Bioconversion of waste glycerol into viscosinamide by Pseudomonas fluorescens DR54 and its activity evaluation

Abstract

Lipopeptides, derived from microorganisms, are promising surface-active compounds known as biosurfactants. However, the high production costs of biosurfactants, associated with expensive culture media and purification processes, limit widespread industrial application. To enhance the sustainability of biosurfactant production, researchers have explored cost-effective substrates. In this study, crude glycerol was evaluated as a promising and economical carbon source in viscosinamide production by Pseudomonas fluorescens DR54. Optimization studies using the Box - Behnken design and response surface methodology were performed. Optimal conditions for viscosinamide production including glycerol 70.8 g/L, leucine 2.7 g/L, phosphate 3.7 g/L, and urea 9.3 g/L were identified. Yield of viscosinamide production, performed under optimal conditions, reached 7.18 ± 0.17 g/L. Preliminary characterization of viscosinamide involved the measurement of surface tension. The critical micelle concentration of lipopeptide was determined to be 5 mg/L. Furthermore, the interactions between the viscosinamide and lipase from Candida rugosa (CRL) were investigated by evaluating the impact of viscosinamide on lipase activity and measuring circular dichroism. It was observed that the α-helicity of CRL increases with increasing viscosinamide concentration, while the random coil structure decreases.

Figure 1

Growth curves of P. fluorescens DR54 growing on MSM supplemented with pure (G1) and crude glycerol (G2–G6). The experiments were performed at 28 °C under constant agitation (180 rpm) using a microplate reader Spark Tecan in triplicate for each type of glycerol.

Figure 2

Glycerol consumption (g/L), growth of P. fluorescens DR54, and viscosinamide production (g/L) performed in MSM supplemented with 40 g/L pure (G1) and waste glycerol (G2–G6) from diverse sources. Experiments were performed at 28 °C and 180 rpm for 120 h.

Figure 3

Pareto chart of standardized effects.

Figure 4

Response surface analysis. Effects of (a) glycerol and leucine, (b) glycerol and phosphate, (c) urea and glycerol, (d) phosphate and leucine, (e) urea and leucine, and (f) urea and phosphate on the concentration of viscosinamide.

Figure 5

Effect of pure viscosinamide concentration (obtained at the optimal conditions) on surface tension. The critical micelle concentration (CMC) was determined from the intersection of regression lines that describe two parts of the curve, below and above the CMC.

Figure 6

Circular dichroism (CD) spectra of lipase from Candida rugosa (1 g/L) alone and in the presence of different concentrations of viscosinamide.