High-cell-density cyclic fed-batch fermentation of a poly(3-hydroxybutyrate)-accumulating thermophile, Chelatococcus sp. strain MW10

Abstract

Different fermentation strategies were employed for the cultivation of a new poly(3-hydroxybutyrate)-accumulating thermophilic bacterium, Chelatococcus sp. strain MW10, with the aim of achieving high-cell-density (HCD) growth and high poly(3-hydroxybutyrate) [poly(3HB)] productivity. Enhanced cultivation was achieved by a cyclic fed-batch fermentation (CFBF) technique (42-liter scale). Maximal poly(3HB) productivity was obtained during the second cycle [16.8 ± 4.2 g poly(3HB)/liter]. At the end of CFBF (265 h), an HCD of up to 115.0 ± 4.3 g cell dry weight/liter was achieved.

FIG. 1.

Fed-batch fermentation for cultivation of the poly(3HB)-accumulating thermophile Chelatococcus sp. MW10. Cells were cultivated in a 2-liter glass bioreactor (Biostat B plus) containing 1.5 liters MSM with glucose as the sole carbon source. The bioreactor was inoculated with a 24-h-grown MSM preculture (4% [vol/vol] inoculum size). Aeration and agitation rates were increased gradually up to 1.25 vvm and 700 rpm, respectively. Cells were grown for 69 h at 50°C and pH 7.3. During the time course of cultivation, samples were withdrawn, and the concentrations of glucose as well as the cell dry weight (CDW) and poly(3HB) content of the cells were determined as described in the text.

FIG. 2.

Cyclic batch fermentation (CBF) for cultivation of Chelatococcus sp. MW10 under thermophilic conditions. Cultivation was conducted in a Biostat UD-30 stirred-tank reactor containing 25 liters MSM with an initial glucose concentration of 50 g/liter as the sole carbon source. The fermentor was inoculated with a 24-h-grown MSM preculture (4% [vol/vol] inoculum size). Culture temperature and pH were controlled at 50°C and at pH 6.7, respectively, during the entire course of the fermentation. Aeration and agitation rates were controlled automatically by adjusting the pO₂ at 20% saturation. Cycling of cultures was operated on a 50-h cycle by withdrawal of 92% of the cultivation medium (black arrows) and refilling with an equal volume of fresh MSM (gray arrows) with glucose (50 g/liter). During the time course of cultivation, samples were withdrawn, and the concentrations of glucose and ammonium as well as the cell dry weight (CDW) and poly(3HB) content of the cells were determined as described in the text.

FIG. 3.

Cyclic fed-batch fermentation (CFBF) for HCD cultivation of the thermophile Chelatococcus sp. MW10. Cultivation was done in a Biostat UD-30 stirred-tank reactor containing 25 liters MSM with glucose as the sole carbon source. The bioreactor was inoculated with a 24-h-grown MSM preculture (4% [vol/vol] inoculum size). (A) Glucose was fed semicontinuously, keeping its concentration higher than 20 g/liter. The glucose feeding solution (30%, wt/vol) was supplemented with MgSO₄ and other nutrients as described in the text. The cycling of cultures was conducted carefully at different intervals, as indicated by black arrows (withdrawal) and gray arrows (refill). Curved gray arrows indicate the semicontinuous feeding. During the time course of cultivation, samples were withdrawn, and the concentrations of glucose and ammonium as well as the cell dry weight (CDW) and poly(3HB) content of the cells were determined as described in the text. (B) Parameters obtained by online monitoring. The optical density was monitored at 850 nm; aeration and agitation rates were controlled automatically by adjusting the pO₂ at 20% saturation; the pH of the medium was controlled at 6.7 using NaOH (5 N); and the fermentation temperature was controlled at 50°C.