Plastic pollution is one of the biggest problems the world is facing these days. To reduce pollution in the future, plastic products must decompose in natural environments in a reasonable period. Polyhydroxyalkanoates (PHAs) are biopolymers that are ubiquitous in nature and excellently biodegradable. Since PHAs have variable properties depending on their molecular structure, they are considered a promising alternative to conventional plastics. PHAs are intracellular carbon and energy storages, which can be efficiently produced by the Gram-negative bacterium Ralstonia eutropha. In particular, oleaginous feedstocks such as plant oils are attractive carbon sources for PHA production with R. eutropha since they are naturally emulsified, have a high carbon content and facilitate high cell density cultivations without large culture dilutions. While the wild-type strain H16 produces the brittle homopolymer polyhydroxybutyrate from plant oils, the engineered strain Re2058/pCB113 accumulates the more flexible copolymer poly(hydroxybutyrate-co-hydroxyhexanoate) (P(HB co HHx)). The main problem in establishing an industrial PHA production is the process cost, which has to compete with low-cost fossil-based plastics. To address this issue, the production process itself must be optimized, either through a comprehensive process understanding or through the ability to monitor the process in real-time, and processes based on alternative low-cost substrates must be developed. For a comprehensive understanding of the applied PHA production process, an untargeted metabolomics analysis of the strains H16 and Re2058/pCB113 during parallel bioreactor cultivations with plant oil was performed. The analysis yielded in two novel findings: (I) Metabolites of the fucose salvage pathway are present, which is a very uncommon pathway in bacteria and (II) the strains accumulate different antioxidants during the cultivations. In a further study, to examine novel possibilities of process monitoring, in-line photon density wave (PDW) spectroscopy was applied for the first time in bacterial cultivations, which enabled an independent quantification of scattering and absorption in real-time. The change of the scattering properties was found to be proportional to the change of the whole cell dry weight (CDW), while the absorption properties were proportional to the change of the residual cell dry weight (CDW minus PHA in g L-1), which consequently allowed to monitor the formation of PHA in real-time. The monitoring of such key process parameters allowed to manually trigger growth, PHA formation and intracellular PHA degradation according to the signal trends. Moreover, feedstock availabilities in Europe were quantitively evaluated, and animal by-product streams were identified as a largely available second-generation feedstock. To expand the possibilities to produce PHA from such low cost substrates, novel bioreactor feeding systems for waste animal fat (WAF) and fat/protein-emulsions (FPEs) were developed, which were required due to the solid nature of the substrates. Fed-batch cultivations with WAF yielded in production of 45 g L-1 P(HB-co-HHx) with a space-time-yield of 0.63 gPHA L 1 h-1. The yield was not altered in scale-down cultivations where the same cultivations strategy was performed, but the cultures experienced short periods of oxygen limitations. The FPE could be used as the sole carbon and nitrogen source in the process. In fed-batch cultivations with the developed pneumatic feeding system, 36 g L-1 P(HB-co-HHx) with a space-time-yield of 0.60 gPHA L-1 h-1 were produced. With proline present in the media, the proline auxotrophic strain Re2058/pCB113 produced a high intracellular PHA content over 70 wt% during the FPE fed-batch cultivation, showing that the plasmid was not lost even without the presence of a selection marker. The obtained results of this thesis should accelerate PHA process development in the future by an enhanced process understanding and the possibility to implement real-time process controllers based on PDW spectroscopy. Furthermore, the validated possibilities to convert WAF-containing feedstocks into PHA should motivate the rendering industry to valorize their by-product streams in this way.
Process development and monitoring of polyhydroxyalkanoate production from oleaginous feedstocks with Ralstonia eutropha
Prozessentwicklung und Monitoring der Polyhydroxyalkanoate-Produktion aus ölhaltigen Rohstoffen mit Ralstonia eutropha
2023
Sonstige
Elektronische Ressource
Englisch
DDC: | 629 |
British Library Online Contents | 2004
|British Library Online Contents | 2004
|British Library Online Contents | 2000
|British Library Online Contents | 2005
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