Team:Edinburgh OG/Demonstrate
Demonstration
The optimisation potential of PHBV production is huge and as a result of our interaction with stakeholders, we developed a number of design requirements for our PHBV production process:
- Use of optimised and innovative processes, such as the use of glucose as carbon source instead of feeding propionic acid into the system.
- Use non-food crops and waste streams.
- Innovative and more cost and environmentally friendly processes for separation and purification.
- Product design.
After incorporating all these design requirements in our final process, the final aim of the team to produce PHBV from waste or industrial by-products. Additionally, we wanted to reduce the impacts of the downstream processing, which is costly environmentally (see our LCA). To demonstrate this we used:
- Bktb/phaCB operon
- Whisky pot ale as raw material
- Phasin and hemolysin secretion system
This section is intended to show how our parts and strategy were implemented and worked as a result of our stakeholders discussions and subsequent design.
PHBV production
Bktb/phaCB operon and Pot ale
We were able to grow our E. coli on media containing M9 salts, 1% glucose, and pot ale. Our negative control contained water instead of pot ale. Our cells grew similar in both types of media but grew lower than our model predicted. However, further experiments need to be done in order to scale and improve the yield by using whisky by-products.
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Figure 1. Six 50 mL cultures of phaCB/Bktb in M9 media and 1% glucose were set up with three containing Pot ale and the other three containing water. Colonies were set up to shake at 37 oC overnight for 64 hours.
We were able to grow our E. coli on media containing M9 salts, 1% glucose, and pot ale. Our negative control contained water instead of pot ale. Our cells grew similar in both types of media but grew lower than our model predicted. However, the pot ale did not negatively affect growth.
Figure 2. OD600 of cells grown in M9 media and 1% glucose with and without pot ale. Cells grown in pot ale and without pot ale similar growth curves.
Next we wanted to show that our cells could produce plastic growing in pot ale. Cells were grown for 64 hours and then had their dry cell weight measured before extraction (Figure 3).
Figure 3. Dry cell weight vs mass of plastic extracted of phaCB-Bktb cells grown in M9 media and 1% glucose with and without pot ale.
PHBV secretion system
PHBV characterisation
Figure 4. Melting temperature ranges of extracted plastic. Cultures grown on pot ale have consistant melting temperature ranges.
We were able to identify by GC-MS two of the main products of PHBV depolymerisation and dehydration by sulphuric acid: crotonic acid and 2-pentenoic acid (Xiang et al., 2016; Braunegg et al., 1978). Demonstrating the presence of PHBV in the processed samples Figure X and Figure X.