Streptomycin production from chitin using Streptomyces griseus
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.