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Digital archive of theses discussed at the University of Pisa


Thesis etd-02222013-182542

Thesis type
Tesi di dottorato di ricerca
Thesis title
Academic discipline
Course of study
tutor Prof. Chiellini, Federica
correlatore Prof. Chiellini, Emo
  • biomaterials
  • Fermentation
  • NIR
  • raw materials
Graduation session start date
The research carried out within the first part of this Doctorate Thesis project is focused on the development of polymeric materials suitable for the development of a nanocarrier system based on a Poly(lactic acid. co – ethyleneglycol) diblock copolymers suitable for biomedical use.
Polymers were synthesized by ring opening polymerization of L-lactide by means of an hydroxyl end-capped methoxy-PEG sample having Mw 5kDa and DPn=115. Polymerization reaction was performed both in bulk and solution (toluene) obtaining 6 g of product characterized by a PLA molar mass equal to 4315 Da and 4186 Da of PLA block respectively. Toxicity evaluated by WST-1 assay and cell morphology test performed on balb/3T3 clone A31 cell line demonstrated how at 1 and 5 mg/ml concentration polymers are not toxic, while toxicity increases at 10 mg/ml. This system was intended for the development of drug delivery systems suitable to be used for different purposes and which could allow overcoming the resistance towards the conventional chemotherapeutic treatments that characterize many solid tumors.
The research carried out within the second part of this Doctorate Thesis project is focused on raw materials studies for fermentation bioprocess improvements.
A high-yield Streptomyces ambofaciens strain has been involved in this study of medium and fermentation process improvement for the production of the macrolide antibiotic spiramycin. Several studies on raw materials used in the fermentation process have been carried out for yield improvement experiments. Activities were focused on starch studies; even if native starch is cheaper than the modified one (enzymatically or chemically digested), the use of modified starch was forced due to the impossibility of enzymatically digesting native starch directly into the industrial fermenter as in the case of laboratory fermentation. Modified starch revealed an improved medium mixing, with an higher dissolved oxygen concentration; this effect was due to the lower viscosity of the medium, which provided better fermentation conditions.
Further studies were undertaken in order to investigate the possibility of modifying trace elements and iron solutions addition, with the aim of simplify the process eliminating contamination risks. The addition of these solutions can occur directly into the medium before sterilization instead of 20 hours after the inoculum through manual sterile connection. The obtained results show how spiramycin yield and impurities content are not affected by this modification.
Corn Oil batches from different suppliers were also studied to improve antibiotic yield: spiramycin productivity, in presence of the new Corn Oil, showed a similar behavior with the standard during the beginning of fermentation. On the contrary, at the end of fermentation productivity it was higher because oil was slowly used by the microorganism.
The last part of the thesis is focused on a NIR (Near Infrared Spectroscopy) approach for the development of a prediction model of yeast extract performances in Corynebacterium diphtheriae fermentation. Yeast extract was chosen as it strictly correlates with Diphtheriae toxin yield. Several yeast extract batches were investigated in term of aminoacid composition and no evident differences were found between high and low performing samples. Preliminary studies, performed with Spectrum Quant+® software for quantitative and qualitative analysis, seem to allow defining a prediction model for outcome yield depending on yeast extract batches used in Diphtheriae fermentation. Even though the promising results, the NIR based method for yeast extract screening and high yield batches identification needs to be further investigated.