Bioprocessing of Marine Chitinous Wastes for the Production of Bioactive Prodigiosin

Abstract

Recently, microbial prodigiosin (PG) has received much attention due to its numerous beneficial applications. The aim of this study was to establish the bioprocessing of marine chitinous wastes (MCWs) for the cost-effective preparation of PG. Of the MCWs, demineralized shrimp shell powders (de-SSP) were found to be a potential source of carbon/nitrogen (C/N) for PG production by bacterial fermentation using Serratia marcescens strains. Further, PG scale-up production was investigated in a 15 L bioreactor system, and the highest yield (6200 mg/L) was achieved during fermentation using 5 L of a novel-designed culture broth that included 1.60% C/N sources (a de-SSP/casein ratio of 7/3), 0.02% K₂SO_4, and 0.05% K₂HPO₄, with an initial pH of 6-7. Fermentation was conducted in the dark at 27.5 °C for 8.0 h. This study was the first to report on the utilization of shrimp wastes for cost-effective, large-scale (5 L/pilot) PG production with high productivity (6200 mg/L) in a short cultivation time. The combination of 0.02% K₂SO₄ and 0.05% K₂HPO₄ was also found to be a novel salt composition that significantly enhanced PG yield. The red compound was purified and confirmed as PG after analyzing its HPLC profile, mass, and UV/vis spectra. The purified PG was then tested for its bioactivities and showed effective anticancer activities, moderated antioxidant activities, and novel anti-NO effects.

Keywords: Serratia marcescens; anti-NO activity; antioxidants; bioreactor; fermentation; prodigiosin; shrimp shells.

Figure 1

Bioproduction of PG by S. marcescens TNU02 by fermentation using various MCWs, such as squid pen powder (SPP), shrimp head powder (SHP), fresh shrimp shell powder (fr-SSP), demineralized crab shell powder (de-CSP), and demineralized shrimp shell powder (de-SSP), as major C/N sources with supplementary casein as a free protein at the ratio of 7.0/3.0. The C/N source (1.60%) was added to a liquid medium of 0.03% K₂HPO₄ and 0.05% CaSO₄. The fermentation was performed for two days at 150 rpm (shaking speed) in the dark at 25℃. The error bars in the figures are standard errors (SE).

Figure 2

The influence of protein sources (a) and de-SSP/casein ratio (b) on PG productivity via fermentation by S. marcescens TNU01. Carbon/nitrogen sources in different proteins were combined with de-SSP at a ratio of 3.0/7.0 (a) and de-SSP was combined with casein in various ratios ranging from 2/8 to 8/2 (b) were used at the concentration of 1.60% in a liquid medium containing 0.03% K₂HPO₄ and 0.05% CaSO₄. Oral casein and oral de-SSP were also fermented for comparison. The fermentation was performed for two days with no light, at 150 rpm and 25 °C. The error bars in the figures are standard errors (SE).

Figure 3

The influence of phosphate salt sources (a), KH₂PO₄ concentration (b), sulfate salt sources (c), K₂SO₄ concentration (d), pH of the liquid medium (e), temperature of fermentation (f), volume of the liquid medium (g), and time course for fermentation (h) on PG yield by S. marcescens TNU01 fermentation. The error bars in the figures are standard errors (SE).

Figure 4

Production of PG in a 15 L bioreactor system and 100 mL flask for different periods of time. The error bars in the figures are standard errors (SE).

Figure 5

The anti-NO activity of prodigiosin (PG) produced via fermentation in this study. The error bars in the figures are standard errors (SE).