Carbon flux to growth or polyhydroxyalkanoate synthesis under microaerophilic conditions is affected by fatty acid chain-length in Pseudomonas putida LS46

Abstract

Economical production of medium-chain length polyhydroxyalkanoates (mcl-PHA) is dependent on efficient cultivation processes. This work describes growth and mcl-PHA synthesis characteristics of Pseudomonas putida LS46 when grown on medium-chain length fatty acids (octanoic acid) and lower-cost long-chain fatty acids (LCFAs, derived from hydrolyzed canola oil) in microaerophilic environments. Growth on octanoic acid ceased when the oxygen uptake rate was limited by the oxygen transfer rate, and mcl-PHA accumulated to 61.9% of the cell dry mass. From LCFAs, production of non-PHA cell mass continued at a rate of 0.36 g L h under oxygen-limited conditions, while mcl-PHA accumulated simultaneously to 31% of the cell dry mass. The titer of non-PHA cell mass from LCFAs at 14 h post-inoculation was double that obtained from octanoic acid in bioreactors operated with identical feeding and aeration conditions. While the productivity for octanoic acid was higher by 14 h, prolonged cultivation on LCFAs achieved similar productivity but with twice the PHA titer. Simultaneous co-feeding of each substrate demonstrated the continued cell growth under microaerophilic conditions characteristic of LCFAs, and the resulting polymer was dominant in C8 monomers. Furthermore, co-feeding resulted in improved PHA titer and volumetric productivity compared to either substrate individually. These results suggest that LCFAs improve growth of P. putida in oxygen-limited environments and could reduce production costs since more non-PHA cell mass, the cellular factories required to produce mcl-PHA and the most oxygen-intensive cellular process, can be produced for a given oxygen transfer rate.

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

“…The low residual levels of carbon and/or NH4 detected in the culture medium at this time (discussed below) may have slowed the growth rate and caused storage of carbon as PHA [34]. This is supported by the observation that Xr production continued until 14 h and then declined to nearly negligible values due to DO limitation, which is consistent with previous work using octanoic acid [26]. Figure 1C shows the profiles of residual carbon NH4, and PO4 3-detected in the culture medium.…”

“…The carbon balance was performed assuming that all consumed carbon could be accounted for through measured CO 2 , PHA biomass (X PHA ), and non-PHA residual cell mass (X r ). The mass balance followed the same approach described previously [26,27].…”

“…Residual octanoic acid in the culture supernatants was measured by GC-FID as previously described [26]. Residual free NH 4 was measured spectrophotometrically at 630 nm via the indophenol blue method (Lachat QuikChem ® Method 10-107-06-1-J) as previously described [27].…”

“…Previously, we have shown that in the production of mcl-PHAs from medium and long chain fatty acids, imposing O 2 limitation on the culture results in a significant redirection of carbon flux toward PHA synthesis [26,27]. However, the aforementioned studies used batch or simple fed-batch strategies as proof-of-concept, and overall productivities were relatively low.…”

“…This is particularly problematic when the substrate exhibits toxicity, and accumulation of substrate can have deleterious consequences [30]. Previously it was found that a fine threshold exists between carbon-limited growth and substrate-induced inhibition when feeding octanoic acid at a predetermined exponential rate (i.e., a proactive feeding approach) [26]. In those experiments, it was observed that carbon limitation was indicated by a rapid and sharp decrease in the off-gas CO 2 concentration, and subsequent rise in the DO signal, when feeding was briefly paused.…”