the whole of this initial inoculum was transferred
into the secondary inoculum in a flask with 70 mL of VIG medium.
After 4 h of growth in the same conditions, this secondary inoculum
was gathered into the tertiary inoculum in a flask with 250 mL of
VIG and was incubated 4 h at 37 ◦C and 100 rpm. Finally, the tertiary
inoculum was added to the batch and fed-batch cultures at 10%
(v/v).
The compositions of the media are summarized in Table 1. Each
marine peptone was used at a level that replaced the Lowry protein
concentration present in the tryptone used for the complex
medium. The preparation and composition of the peptones solutions
from shark and thornback ray was described in a previous
work [25]. Moreover, in order to reduce the hyaluronidase activity
releases by S. zooepidemicus we have included 15 mg L−1 of
polystyrene (Mw = 990 kDa, Sigma) in the culture media. In all cases,
the initial pH was adjusted to 6.7 and the media were sterilised
at 121 ◦C for 15 min. Cultures were carried out in duplicate using
a glass 2 L-bioreactor with a working volume of 1.8 L. All fermentations
were performed without aeration at 37 ◦C, with agitation
of 500 rpm and the pH was automatically controlled with sterile
5 M NaOH. In the fed-batch cultures, the reducing sugars profiles
were always maintained above 10 g L−1 by repeated added of sterile
glucose solution of 500 g L−1.