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.
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.
Our liquid cultures show the growth of our Bktb strain in M9 liquid media. The growth curve we got from the pot ale supplemented media is lower than our model predicted and did not increase cell growth greater than media using water. However, from this data, we can not say that pot ale supplemented media increases 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. Mass of plastic extracted of phaCB-Bktb cells grown in M9 media liquid media.
The extracted plastic we obtained ranged from 11.9 mg to 17.3 mg. The largest and smallest amount came from cultures grown in pot ale.
When the cultures were dried out, it seemed that the cultures grown on pot ale had larger cell pellets but may have been larger because of the left-over sediment in the pot ale. We would expect similar dry cell mass from each culture based off the similar OD600 readings.
PHBV characterisation
The melting temperatures of the pot ale and water cultures (Figure 4) have melting temperatures all lower than PHB which has a typical melting temperature of 170-180 oC. The more PHV incorporated into PHBV would lower the melting temperature. By using pot ale we obtained a range of 160-168 oC. However, our cultures grown in M9 media without pot ale had a wider range of 158-170 oC.
Figure 4. Melting temperature of plastic extracted of phaCB-Bktb cells grown in M9 media liquid media.
However, we can see when 8 mM propanoic acid is added we see an even lower melting temperature (Table 1).
Table 1. Melting Temperature Measurement of PHB, PHBV, phaCAB, and phaCAB/bktb and phaCB/bktb produced plastic grown in media with 8mM propanoic acid.
After analysis of our plastic produced by our bktb-cells in pot ale, 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).
Figure 5. Sample MW: PHBV production in M9 media without propionic acid added. Figure 6. Sample 2P: PHBV production in M9 media and pot ale without propionic acid added. 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 5 and Figure 6.
PHBV secretion system
As mentioned in the integrated human practice section. the downstream processing of PHA is one of the big problem in current industry. Secretion of PHA bioplastic could reduce the cost of obtaining the PHB and in theory would allow us to establish a sustainable PHA producing E. coli culture. In order to tackle this issue, we have developed a PHA secretion project leaded by Owen and Qihui, which related to the phasin autoregulation system, to improve the PHA secretion. Through the newly developed contruct: PhaR-Phasin-HlyA, an increased amount of PHB production and secretion were observed.