Characterization of the Metabolic Response of Streptomyces clavuligerus to Shear Stress in Stirred Tanks and Single-Use 2D Rocking Motion Bioreactors for Clavulanic Acid Production

Abstract

Streptomyces clavuligerus is a gram-positive filamentous bacterium notable for producing clavulanic acid (CA), an inhibitor of β-lactamase enzymes, which confers resistance to bacteria against several antibiotics. Here we present a comparative analysis of the morphological and metabolic response of S. clavuligerus linked to the CA production under low and high shear stress conditions in a 2D rocking-motion single-use bioreactor (CELL-tainer ^®) and stirred tank bioreactor (STR), respectively. The CELL-tainer^® guarantees high turbulence and enhanced volumetric mass transfer at low shear stress, which (in contrast to bubble columns) allows the investigation of the impact of shear stress without oxygen limitation. The results indicate that high shear forces do not compromise the viability of S. clavuligerus cells; even higher specific growth rate, biomass, and specific CA production rate were observed in the STR. Under low shear forces in the CELL-tainer^® the mycelial diameter increased considerably (average diameter 2.27 in CELL-tainer^® vs. 1.44 µm in STR). This suggests that CA production may be affected by a lower surface-to-volume ratio which would lead to lower diffusion and transport of nutrients, oxygen, and product. The present study shows that there is a strong correlation between macromorphology and CA production, which should be an important aspect to consider in industrial production of CA.

Keywords: Streptomyces; antibiotic; bioreactors; clavulanic acid; fed-batch; morphology; oxygen transfer; shear stress; single-use; stirred tank.

Figure 1

Time course of biomass in stirred tank bioreactor (STR, triangles) and CELL-tainer^® (CT, circles) in fed-batch cultivations of Streptomyces clavuligerus.

Figure 2

Morphological response of S. clavuligerus in STR (triangles) and CT (circles) bioreactors: (a) Mycelia in STR at 22 h; (b) Mycelia in STR at 86 h; (c) Mycelia in CT at 23 h. (d) Mycelia in CT at 87 h; (e) Time course of mycelial diameter in STR (blue) and CT (red).

Figure 3

Dynamic profiles of oxygen in liquid and exhaust gas in fed-batch cultivations of S. clavuligerus: (a) Dissolved oxygen (DO) in STR (blue) and CT (red) cultivations; (b) Respiratory Quotient (RQ) in STR (blue) and CT (red) cultivations.

Figure 4

Time courses of substrates in the culture media for fed-batch cultivations of S. clavuligerus. Glycerol in STR (triangles) and CT (circles), glutamate in STR (squares) and CT (diamonds).

Figure 5

Time courses of clavulanic acid (CA) and phosphate in fed-batch cultivations of S. clavuligerus. CA in STR (triangles) and CT (circles), phosphate in STR (squares) and CT (diamonds).

Figure 6

Time courses of succinate and pyruvate accumulations in fed-batch cultivations of S. clavuligerus. Succinate in STR (triangles) and CT (circles), pyruvate in STR (squares) and CT (diamonds).