The production of streptomycin using Streptomyces griseus using two types of
chitin as a substrate was studied using a variety of fermentation techniques.
Commercial chitin was obtained (Sigma) and comprised chemically purified crab
shell. Pre-fermented chitin was the solid product from the lactic acid
fermentation of shrimp waste using Lactobacillus paracasei A3. Bioassay,
HPLC and FTIR methods were developed during this project for the
quantification of streptomycin both in liquid phase and adsorbed on solid chitin
surfaces.
Shake flask experiments were carried out to determine basic production kinetics,
as well as to establish if commercial and pre-fermented chitins produced
different quantities of streptomycin. Shake flasks were also used to evaluate any
effect of chitin concentration on streptomycin production. A range of submerged
fermentations were undertaken in a standard 2 L bioreactor fitted with Rushton
Turbines, at chitin concentrations from 0.4 %w/v to 10 %w/v, to study the effect
on streptomycin yield. At concentrations of 5 %w/v and over, it was necessary to
use an alternative, V-shaped agitator, as the Rushton Turbines did not provide
adequate mixing. The V-shaped agitator was designed and produced as part of
this project. The submerged fermenter was also used to determine if the re-use of
.chitin remaining post-fermentation was possible.
A solid state fermentation packed bed bioreactor was also developed, with a
recycle loop for produced liquor. Four experiments evaluated the use of
commercial and pre-fermented chitins, and different liquid media used for
inoculation. In order to encompass the advantages of submerged and solid state
fermentations, a vertical basket reactor was designed and manufactured, which
used gentle fluidisation for the agitation of chitin particles contained inside the
basket.Shake flask experimentation showed that pre-fermented chitin produced
approximately 3 times the streptomycin yield than that of commercial chitin.
Both systems reached a maximum liquid phase yield after 8 days of
fermentation. Maximum streptomycin yields were obtained at a chitin
concentration of 10 %w/v.
The total streptomycin yields from submerged fermentation were fairly
consistent over the range of chitin concentrations used. The amount of
streptomycin adsorbed on the chitin surface, however, increased with increasing
chitin concentration. Total streptomycin yields varied from 2 to 3.5 mglL. The
re-use of chitin remaining post-fermentation was found to be possible in two
series of three experiments. In both cases (at approximately 7.5 %w/v and 10
%w/v chitin) the lag phase and time to reach maximum biomass concentration
decreased. Particle size analysis and mathematical modelling suggest that this is
due to increasing specific surface of chitin particles during the course of
fermentation.
Both shake flask and submerged fermentation showed a bioassay inhibition peak
in the tropophase, removable using 2 kDa membrane filters. Although it was not
possible to determine the exact nature of the inhibiting component(s),
streptomycin was eliminated through FTIR. A study of chitosan oligomers
showed that short chain oligosaccharides inhibit Bacillus subtilis in a similar
manner to streptomycin.
Solid state fermentation using a salts solution liquid medium, with intermittent
aeration and recirculation proved to be the most effective, giving a streptomycin
yield of 3.8 mg/L. The vertical basket reactor obtained higher streptomycin
yields of 4.6 mg/L. Post-fermentation washing with pH 3 buffer was also
successfully used in this fermenter for the in-situ extraction of streptomycin,
before the addition of fresh sterile liquid medium and fermentation re-start
