Process development for fed-batch production of medium-chain-length poly(3-hydroxyalkanoate) from decanoic acid
β-oxidation inhibition , Decanoic Acid , MCL-PHA , Poly-hydroxydecanoate , Fed-batch , Fermentation , Medium-chain-length Poly-3-hydroxyalkanoate , Pseudomonas putida
The main goal of the thesis is to develop strategies for the fed-batch production of medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) by Pseudomonas putida KT2440 from decanoic acid (DA) co-feeding with glucose (G). Since decanoic acid is poorly water soluble and a solid at room temperature, two methods were tested to maintain it as a liquid by either mixing it with acetic acid (AA) or heating it to feed into the bioreactor. Exponentially feeding the carbon substrates at a ratio of 3:3:4 of DA:AA:G at a specific growth rate (µ) of 0.15 h-1 resulted in 26 g L-1 biomass containing 57% PHA. The final PHA composition was 74 mol% 3-hydroxydecanoate (3HD), 14 mol% 3-hydroxyoctanoate (3HO) and 12 mol% 3-hydroxyhexanoate (3HHx). As the ratio of DA:AA:G increased from 3:3:4 to 5:1:4, 50% higher biomass and 73% higher PHA were obtained. An even higher biomass concentration of 75 g L-1 containing 74% PHA was achieved by applying the same specific growth rate followed by constant substrate feeding strategy. Heating decanoic acid without acetic acid was performed as a control experiment at feeding ratio of DA:G (1:1) resulting in a final higher biomass of 41 g L-1 but containing only 58% PHA. A fadB and fadA knockout mutant of P. putida KT2440 named P. putida DBA-F1was constructed to increase the fraction of the dominant monomer (3HD) in PHA by limiting the fatty acid β-oxidation activity. Using a feeding ratio of decanoic acid and acetic acid (DAA) to glucose (G) (6:4) at specific growth rate of 0.15 h-1, significantly different monomeric composition (97% 3HD:3% 3HO) but a lower biomass (8.5 g L-1) and PHA concentrations (3.5 g L-1) were obtained compared to the wild type. The performance could be improved at a feeding ratio of DAA:G of 2:8, leading to 18 g L-1 biomass containing 100% poly(3-hydroxydecanoate) (PHD). MCL-PHA with enriched 3HD monomers could also be obtained using acrylic acid (Aa) as a chemical inhibitor of the β-oxidation pathway by P. putida KT2440. At a ratio of DAA:G:Aa (6:4:0.2), 21 g L-1 MCL-PHA with 84 mol% 3HD was produced.