Abstract
This study aimed to develop methods to improve PHA production from selected volatile fatty acids (VFAs) and to improve the understanding of the PHA production process by pure culture bacterial fermentation using Cupriavidus necator. Optimisation strategies involved the following investigations using shake flasks and 5 litre based batch fermentations:Shake flask investigations determined that the temperature of 30oC and a nutrient medium, resulted in the highest growth of bacteria. A feeding strategy of the substrate (VFAs – acetic acid and butyric acid) was developed to avoid inhibition by the substrate and the alkaline buffer. The results established that continuous feeding of VFAs based on maintaining optimum pH around 7 resulted in enhanced PHA yields by almost 2-fold when compared to single pulse feeding of acetic and butyric acids (at 1, 2, 3, 4 and 5 g/l VFA concentration).
A novel application of a capacitance probe was demonstrated to be able to monitor maximum PHA accumulation in-situ and in real-time, so as to prevent product and substrate loss (acetic and butyric acids, by a maximum value of 12 g/l and 20 g/l respectively), and to increase PHA process understanding and fermentation kinetics. The dielectric spectroscopy probe was able to correlate very well (R2 = 0.862) with PHA off-line measurements when operated in dual frequency mode and was able to establish the optimum PHA harvesting time, which would have resulted in improved process economics and environmental performance.
Shake flask experiments were conducted to investigate the addition of trace metals (zinc, manganese, boric acid, cobalt, nickel and sodium molybdate), copper and sodium chloride in nutrient media and its effects on bacteria growth. NaCl contributed to the greatest enhancement in the early growth of bacteria and therefore fed batch fermentations using 0, 3.5, 6.5, 9, 12 and 15 g/l of additional NaCl concentrations were evaluated. The 9 g/l NaCl concentration showed the highest PHA production of 5.33 g/l and also caused PHA accumulation to occur earlier by 2 h than the control. The capacitance probe also helped visualise and understand the bacterial growth and PHA accumulation profile.
The novel use of low cost digestates based media was demonstrated. Results demonstrated that possibly due to the nutrients/trace elements in a digestate (from food wastes and wheat feed) the PHA accumulation was enhanced by 3-fold (to 12.29 g/l); with a resulting highest ever reported PHA accumulation of 90% for C. necator. C: N: K: P: S ratios for this digestate based fermentation were found to be for growth 761: 31: 1: 3.5: 1.9 and for PHA accumulation 1132: 11: 3: 1.7: 1. Digestates use within biotechnology and biorefining specifically for bacterial applications could provide another alternative route to digestate disposal that may lead to valuable end products.
All the above evaluations represented novelty and have delivered significant process optimisation for PHA production from VFAs.
| Date of Award | Oct 2014 |
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| Original language | English |
| Supervisor | Sandra Esteves (Supervisor), Richard Dinsdale (Supervisor) & Alan Guwy (Supervisor) |