Novel Polyhydroxyalkanoate blends: their characterisation and possible applications

Polyhydroxyalkanoates (PHAs) are polyesters consisting of 3-hydroxyalkanoic acids synthesised by numerous bacteria as storage compounds, in the presence of excess carbon, under nutrient limiting conditions. PHAs are biodegradable and biocompatible polymers that exhibit a variety of properties ranging from being thermoplastic to elastomeric in nature. For the first part of this study, the production of PHA in Bacillus cereus SPV and Pseudomonas mendocina was investigated. Nutrient limitations play a major role in PHA production hence, studies were carried out on the effect of nitrogen, potassium and magnesium limitations on the short chain length PHA accumulation by B. cereus SPV. The organism was grown in the Kannan and Rehacek medium using sucrose as the carbon source and accumulated PHA with a maximum yield of 38.0% dcw was observed in the shaken flask cultures. The study was continued with batch fermentation studies in 2 litre fermenters and an enhanced PHA yield was observed with an optimum yield of 44.6% dcw. Further, an enrichment media (MEM media) for B. cereus SPV was modified, with three simultaneous nutrient limitations for the production of PHA. A further improved yield of the polymer (52.64% dcw) was observed in this novel media. Chemical analysis of the extracted polymer was carried out using NMR and it was found that the organism accumulated the homopolymer of P(3HB).

When Pseudomonas mendocina was grown in the mineral salt media (MSM) with a sodium octanoate as sole carbon source, medium chain length PHA accumulation was observed with a maximum polymer yield of 29.43% dcw in shaken flask cultures. The study continued with batch fermentation studies on the production of the PHAs, which was carried out using 2 Litre fermenters and an improved yield of the polymer (33.5 wt% dcw) was noted. Fed batch fermentation was also explored and a further increase in polymer accumulation was obtained, giving a maximum yield of 37.09% dcw. Chemical analysis of the polymer using NMR proved that the organism accumulated a homopolymer of Poly(3-hydroxyoctanoate) P(3HO), a rare occurrence. P. mendocina was also grown in MSM media with sucrose as carbon source and PHA accumulation was observed with a yield of 27.19% dcw. The polymer was structurally analysed by NMRand identified as the homopolymer of P(3HB).This is the first time that an absolute homopolymer of P(3HB) has been produced by P.mendocina using sucrose as the carbon source.

A detailed study on the effects of different extraction methods on the yield of the PHAs was carried out. Among different extraction methods used for PHA extraction the dispersion method gave the highest PHA yield of 30% dcw. The chloroformextraction showed the polymer yield of 28% dcw.The soxhlet extraction, gave the lowest yield of 12% dcw.A novel PHA recovery and purification method based on the osmotic and detergent based lysis and purification was also successfully developed. Higher purity (25%)of the extracted PHA compared to dispersion method, was confirmed by GC analysis.

The blending of the flexible and soft P(3HO) extracted from P.medochina with the brittle and stiff P(3HB) from Bacills cereusSPV was carried out in two ratios, 5:1 and 1:5. The thermal analysis of P(3HB) showed that the polymer sample had a high melting temperature Tm of 167.39°C, a glass transition temperature, Tg of 2.43°C and a crystallisation temperature Tc value of 54.33°C. The thermal analysis of P(3HO) showed that the polymer exhibited low melting temperature of 50.36°C, a Tg of -32.86°C), and no Tc value. The P(3HB)/P(3HO) 5:1 blend showed two melting temperature 164.91°C and 157.22 °C, single lower glass transition temperature of 5.84°C and raised Tc of 69.58°C as compared to neat P(3HB).The P(3HO)/P(3HB) 5:1 blend on the other hand higher melting temperature of (164.85°C), lower glass transition temperature of -36.990C as compared to P(3HO) and no Tc was observed.

The P(3HO)/P(3HB) (5:1) blend showed an Young modulus value of 37 MPa with a tensile strength of 1.5 MPa and elongation to break of 160%. Increasing the amount of P(3HB) as in the case of P(3HB)/P(3HO) (5:1) increased the Young modulus value to 4.99 MPa indicating an increase in the stiffness. The percentage of elongation of the film was reduced to just 35.81%. The incorporation of P(3HB) into the biopolymer matrix of P(3HO) or P(3HO) into a predominantly P(3HB) matrix thus resulted in a change in the mechanical properties of the neat PHAs. P(3HB) served the purpose of increasing the tensile strength of the blend, whereas P(3HO) served the purpose of increasing the elasticity of the material. The flexible and strong nature of the P(3HO)/P(3HB) (5:1) blend would make it suitable for a variety of application including the preparation of nerve conduits.

The water contact angle value for neat P(3HB) film was 70.37o and for P(3HO) was 99.94o. In the case of blend films P(3HO)/P(3HB) (5:1) and P(3HB)/P(3HO)(5:1), the θH2O was 90.39o and 80.0o respectively. The water contact angle studies showed that both the neat and blend PHAs are hydrophobic in nature.

Both the neat and blend films were able to support the attachment, growth and proliferation of the HaCaT cells. However, biocompatibility was better for the P(3HO)/P(3HB) 5:1 film and the topological features of this film led to improved cell attachment and proliferation. SEM analysis confirmed that the HaCaT cells had been able to grow and mature faster on the P(3HO)/P(3HB) (5:1) blendfilm. In conclusion, this work has led to the development of novel PHA blends with new properties, which can be exploited in a variety of applications including nerve tissue engineering.