Development of High Cell Density Cultivation Strategies for Improved Medium Chain Length Polyhydroxyalkanoate Productivity Using Pseudomonas putida LS46

Abstract

High cell density (HCD) fed-batch cultures are widely perceived as a requisite for high-productivity polyhydroxyalkanoate (PHA) cultivation processes. In this work, a reactive pulse feed strategy (based on real-time CO2 or dissolved oxygen (DO) measurements as feedback variables) was used to control an oxygen-limited fed-batch process for improved productivity of medium chain length (mcl-) PHAs synthesized by Pseudomonas putida LS46. Despite the onset of oxygen limitation half-way through the process (14 h post inoculation), 28.8 ± 3.9 g L−1 total biomass (with PHA content up to 61 ± 8% cell dry mass) was reliably achieved within 27 h using octanoic acid as the carbon source in a bench-scale (7 L) bioreactor operated under atmospheric conditions. This resulted in a final volumetric productivity of 0.66 ± 0.14 g L−1 h−1. Delivering carbon to the bioreactor as a continuous drip feed process (a proactive feeding strategy compared to pulse feeding) made little difference on the final volumetric productivity of 0.60 ± 0.04 g L−1 h−1. However, the drip feed strategy favored production of non-PHA residual biomass during the growth phase, while pulse feeding favored a higher rate of mcl-PHA synthesis and yield during the storage phase. Overall, it was shown that the inherent O2-limitation brought about by HCD cultures can be used as a simple and effective control strategy for mcl-PHA synthesis from fatty acids. Furthermore, the pulse feed strategy appears to be a relatively easy and reliable method for rapid optimization of fed-batch processes, particularly when using toxic substrates like octanoic acid.

Engineering Native and Synthetic Pathways in Pseudomonas putida for the Production of Tailored Polyhydroxyalkanoates

“…Total biomass of 28.8 g L −1 and a PHA content of 61% (w/w) were achieved after 27 h using octanoic acid as the carbon source in a bioreactor operated under atmospheric conditions—resulting in final volumetric productivity of 0.66 g PHA L −1 h −1 . [ 157 ] As such, high‐cell‐density cultivation processes, combined with the synthetic biology, metabolic engineering, and systems biology approaches—as summarized in the previous sections of this review—can help to overcome the long‐standing problem of low PHA titers. This aspect is particularly important for producing novel biopolymers (which are typically accumulated in limited amounts) to achieve an economically competitive industrial scale.…”

Evaluation of 3D-Printing Scaffold Fabrication on Biosynthetic Medium-Chain-Length Polyhydroxyalkanoate Terpolyester as Biomaterial-Ink

“…The polyester biosynthesized by Pseudomonas putida ATCC47054 was isolated and purified to determine the PHA content using GC analysis and the PHA monomeric composition by 1 H-NMR, as listed in Table 1. The PHA productivity and product yield after 60-h cultivation were 0.067 g/L/h and 0.05 g PHA/g substrates, respectively, which is relatively low compared to other reports [27]. The chemical functional group analysis was performed using Fourier Transform Infrared Spectrophotometer (FTIR), as shown in Fig- ure 4.…”

Optimized pulse‐feeding fed‐batch fermentation for enhanced lignin to polyhydroxyalkanoate transformation

“…Previous works have reported that the pulse feeding scheme was preferred than the continuous constant-feed technique yielding higher PHA accumulation. [23][24][25] The pulse feed strategy was better at cycling between carbon excess and carbon-limited conditions during fedbatch, hence enhancing PHA synthesis. In addition, during pulsefeeding condition, cell growth was less restricted compared to continuous feeding condition.…”