Biosynthesis of polyhydroxyalkanoates and its medical applications

Rai, R. 2010. Biosynthesis of polyhydroxyalkanoates and its medical applications. PhD thesis University of Westminster School of Life Sciences

TitleBiosynthesis of polyhydroxyalkanoates and its medical applications
TypePhD thesis
AuthorsRai, R.
Abstract

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.

Year2010
FileRanjana_RAI.pdf
Publication dates
Completed2010

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