Resumen de Study for poly[(R)-3-hydroxybutyrate] production by bacterial strains and its modification for drug delivery systems
Alejandra María Rodríguez Contreras
Nowadays biopolymers are a good alternative to petrochemical polymers. One of these biopolymers is Poly[(R)-3- Hydroxybutyrate] (PHB) which is produced from renewable resources by various microorganisms and, after its use it can be composted. It has different applications as bulk material in packing films, containers or paper coatings. Due to its excellent biocompatibility and biodegradability, PHB is a potential material for applications in drug delivery. It is therefore important to continue investigating the different strategies to ensure the competitiveness between these biopolymers and petroleumbased polymers. The first part of the thesis is mainly focused on finding a new PHB-producing bacterium capable of developing and producing a significant concentration of biopolymer using one of the conventional medium for PHB industrial production. Therefore, four water and mud samples were taken from four Bolivian salty lakes and the microorganisms were isolated in order to detect the best PHB-producing bacterial strain. A bacterium identified as a new strain of the genus Bacillus was selected for polymer production studies. This microorganism was tested under fed-batch fermentations. The results showed a high specific growth rate, and a percentage of 70% PHB in cell dry mass , with limitation in nitrogen source. The strain not only grew properly in the industrial condition proposed, but it also produced and accumulated a larger content of PHB than ever reached before for its genus. The strain was deposited in the Spanish Type Culture Collection (CECT), as Bacillus megaterium uyuni S29. The characterization of the resultant biopolymers concluded that the strain synthesized PHB homopolymer with two main molecular masses around 600 and 125 kDa with polydispersity index of 1.2 and 1.5 respectively. Moreover, the thermal analyses of the biomaterial showed different properties compared to the ones of common PHB, enlarging the application possibilities to this biopolyester. After obtaining the polymer from the bacteria, the second part was focused on its transformation. A sustainable degradation process set out for an easy industrial scale application was proposed in the second part of this thesis. A copolymer from PHA family, poly[3-hydroxybutyrate-co-4-hydroxybutyrate] , was used to carry out an enzymatic degradation of the material. Two commercially available lipases were used to decrease the polymer molecular mass to oligomers between 5 and 1 kDa. It was the first time that these triglyceride lipases were used for this type of application and due to the positive results one of them was used for degradation of PHB. The results confirmed the enzymatic reaction of the used lipase with PHB and showed a controlled decrease of the molecular mass from 300 kDa to 4 kDa. In the third part of the study, the application of the PHB as a drug delivery system was analyzed. Doxycycline is a well known broad-spectrum antibiotic but there are some concerns over its possible side effects. In order to avoid this problem and reach the infection with an effective drug concentration, protection and controlled delivery of doxycycline are desirable. Thus, different drug-entrapment and emulsification methods were studied in order to obtain optimal doxycycline -loaded PHB micro- and nano-particles. The results showed that the combination of ultrasounds with high speed stirring in the preparation of double emulsion is highly effective in obtaining doxycycline -loaded PHB particles with high drug loading and entrapment efficiency, and with great method efficiency. Furthermore, the qualitative antibacterial activity found in different formulations show that these particles are good candidates for drug delivery systems. These best methods were used with the PHAs produced throughout the thesis in order to confirm their suitability for different PHAs and to study the possibility of improving the results of drug entrapment and method efficiency.
