Biosynthesis of polyhydroxyalkanoates and its medical applications
Creators: name: Rai, Ridesh
Publisher: name: University of Westminster
Year: 2010

description: Polyhydroxyalkanoates are polyesters of 3-hydroxyalkanoic acids produced by numerous Gram positive and Gram negative bacteria under nutrient limiting conditions. Once extracted, the PHAs exhibit a variety of properties from thermoplastic to flexible elastomeric nature. Biodegradability and bicompatibility of PHAs are also well established. Owing to these properties PHAs are increasingly attracting interest for commercial exploitation in various agricultural, industrial and particularly medical applications. The main aim of this study was to investigate PHA production in microorganisms and utilise the PHA produced for medical applications. Nutrient limitations play a pivotol role in PHA production, hence studies were carried out on the effects of nitrogen, phosphorous, potassium and sulphate limitations on the short chain length, scl-PHA (C3-C5 carbon chain length) accumulation by B. cereus SPV. The organism accumulated P(3HB) under nitrogen (38 % dcw), sulphur (13.15 % dcw), phosphorous (33.33 % dcw) and P(3HB-co-3HV) under potassium limitations (13.4 % dcw). Studies were also carried out on the production of medium chain length, mcl-PHAs (C6-C14 carbon chain length) using five different Pseudomonas sp., P. aeruginosa, P. putida, P. fluorescens, P. oleovorans and P. mendocina. GC-MS analysis confirmed the presence of the monomer 3-hydroxyoctanoate in the polymer extracted from P. putida. However the result could not be confirmed with NMR. P. oleovorans was shown to accumulate copolymers of 3HO and 3-hydroxyhexanoate. P. aeruginosa accumulated a novel copolymer containing the monomers 3-hydroxyoctanoate and 2-hydroxydodecanoate, P(3HO-co-2HDD) when grown in octanoate. Occurrence of monomers other than 3-hydroxyacid is rare hence accumulation of P(3HO-co-2HDD) by the organism was interesting. P. fluorescens did not accumulate any polymer. P. mendocina was the main organism that was focussed on for mcl-PHA production because of it being relatively unexplored amongst other Pseudomonas producers. The organism showed interesting mcl-PHA biosynthetic capability. It accumulated a homopolymer of P(3HO) (31.3 % dcw) when grown in octanoate. This is the first time that an absolute homopolymer of P(3HO) has been produced. P. mendocina also accumulated a copolymer of P(3HB-co-3HO) when grown in sucrose. Such copolymers containing both scl and mcl monomers occur rarely. A detailed study on the effects of different extraction methods on the yield, molecular weight, thermal properties and lipopolysaccharide (LPS) content of P(3HO) was carried out. An optimised extraction method using a dispersion of hypochlorite and chloroform combined with an optimised polymer purification was found to extract P(3HO) efficiently and also reduce the amount of LPS to an FDA approved level of 0.35 EU/mL. The homopolymer P(3HO) was also studied as a potential biomaterial for medical applications. The polymer was fabricated into neat P(3HO) films to be used a biomaterial for pericardial patch application. Bioactive nanobioglass particles (n-BG) of the type 45S5 Bioglass® were incorporated as a filler in the polymer matrix to form P(3HO)/n-BG composite films. The P(3HO)/n-BG composite films were to be used as a multifunctional wound dressing which would act both as a biomaterial for skin regeneration and also provide a haemostatic effect. Yes, the P(3HO) in combination with n-BG i.e. the P(3HO)/n-BG composite film was studied for wound dressing. This has been pointed out in Chapter 5: sections 5.1.2. and 5.3.5 The n-BG was found to accelerate blood clotting time confirming the haemostatic effect. The roughness, wettability and Young’s modulus of the neat films was increased by the incorporation of the n-BG. Both the neat and composite films were flexible and elastomeric in nature. The E value of the 5 wt% neat film (1.4 MPa) was suitable for its use as a pericardial patch material. The flexible nature of the P(3HO)/n-BG composite film would make it suitable for applications in difficult contours of the body. Both the neat and composite films were able to support the attachment, growth and proliferation of the HaCaT cells. However, biocompatibility was improved for the P(3HO)/n-BG composites. In vitro degradation studies revealed the films both neat and composite underwent hydrolytic degradation which started at the surface and that aged with time. Modification of P(3HO) was also carried out. This was done by exposing P(3HO) to UV rays, incorporating n-BG into the UV treated polymer matrix to form composites by and blending the flexible elastomeric P(3HO) with the hard and brittle Poly-3-hydroxybutyrate, P(3HB) produced from B. cereus SPV and incorporating n-BG into this blend polymer matrix. UV treatment of the polymer increases it hydrophilicity and surface roughness. However, UV treatment also caused P(3HO) chain scissions which increased its surface roughness and also caused cross linking of polymer chains. Both the UV P(3HO) neat and UV P(3HO)/n-BG composite (neat and composite films) made from UV treated P(3HO) showed improved biocompatibility over the non UV treated polymer counterparts for the seeded HaCaT cells. The films showed signs of polymer ageing and underwent slow hydrolytic degradation possibly because of the cross linked P(3HO) chains. For the blend films the surface properties was greatly affected by the amount of P(3HB) incorporated. The roughness was higher for the blend film containing higher wt% of P(3HB). The roughness was further increased with the incorporation of n-BG into this blend matrix. The stiffness of P(3HO) increased due to the incorporation of P(3HB). In vitro degradation studies revealed that the fabricated blend and composite blend films underwent hydrolytic degradation.