POC 03: Improving the economics of poly-B-hydroxybutyrate (PHB) production based on polymerisation of secreted monomers.

Project lead

David Leak

Institute

University of Bath

Poly-β-hydroxybutyrate is a biodegradable plastic produced as an intracellular polymer by some bacteria. While it has many potential uses, it is too expensive to produce for general application and is currently only used in specialist applications (eg medical).

Aims

Poly-β-hydroxybutryate (PHB) is a useful biopolymer with a limited current market due to the production economics. It is produced intracellularly at up to 60% (w/w) of the host organism. Our aim was to show that it is possible to make PHB by producing the monomeric R-β-hydroxybutyrate in high titres as a secreted product and polymerise it chemically. This would allow production from a wider range of substrates and improve the economics of production.

Outcomes

Integration (3 partners) of this project was impacted by Covid 19, meaning that separate parts were done in isolation, effectively in reverse order! Thus, the extraction, purification and polymerisation of monomeric R-β-hydroxybutyrate was initially demonstrated by using a simulated media. Once a hydrolysed organic fraction of MSW (OFMSW) was available this was introduced into the simulated media and the purification repeated. This showed that a yellow pigmented material derived from the OFMSW was getting through the purification process. There was not sufficient time to improve the purification, or to assess how this affected polymerisation.

The recombinant strain construction and evaluation progressed in parallel. While the ability to utilise both xylose and glucose as substrates was demonstrated in a simulated media, confirmation of growth on the OFMSW was only obtained at the end of the project. Gene and operon design were straightforward but the host strain had to be changed from that originally envisaged, due to the inability to reproduce a published transformation procedure. This required some modification of the genetic construction and a switch to a transformable host organism. Low levels of secreted hydroxybutyrate were observed, but there was insufficient time for further (flux/secretion) optimisation.

Generation of fermentable carbohydrates from the OFMSW was delayed; only two early samples with low monomeric carbohydrate content were available for evaluation towards the end of the project. They were used as described above, but subsequent improvement of the hydrolysis process generated streams with >10% (w/v) monomers, sufficient for commercially relevant batch fermentation.

Impact

We have demonstrated that hexose and pentose monomers from OFMSW can be fermented by a recombinant Parageobacillus sp to produce R-β-hydroxybutyrate which can be recovered, purified and chemically polymerised. Bottlenecks in flux or secretion in the recombinant strain need to be resolved before the commercial potential can be fully evaluated.

Industrial partners: Black Jumper Ltd; Activatec