Putting cells under pressure: A simple and efficient way to enhance the productivity of medium‐chain‐length polyhydroxyalkanoate in processes with Pseudomonas putida KT2440

Abstract

The success of bioprocess implementation relies on the ability to achieve high volumetric productivities and requires working with high-cell-density cultivations. Elevated atmospheric pressure might constitute a promising tool for enhancing the oxygen transfer rate (OTR), the major growth-limiting factor for such cultivations. However, elevated pressure and its effects on the cellular environment also represent a potential source of stress for bacteria and may have negative effects on product formation. In order to determine whether elevated pressure can be applied for enhancing productivity in the case of medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida KT2440, the impact of a pressure of 7 bar on the cell physiology was assessed. It was established that cell growth was not inhibited by this pressure if dissolved oxygen tension (DOT) and dissolved carbon dioxide tension (DCT) were kept below ∼30 and ∼90 mg L(-1), respectively. Remarkably, a little increase of mcl-PHA volumetric productivity was observed under elevated pressure. Furthermore, the effect of DCT, which can reach substantial levels during high-cell-density processes run under elevated pressure, was investigated on cell physiology. A negative effect on product formation could be dismissed since no significant reduction of mcl-PHA content occurred up to a DCT of ∼540 mg L(-1). However, specific growth rate exhibited a significant decrease, indicating that successful high-cell-density processes under elevated pressure would be restricted to chemostats with low dilution rates and fed-batches with a small growth rate imposed during the final part. This study revealed that elevated pressure is an adequate and efficient way to enhance OTR and mcl-PHA productivity. We estimate that the oxygen provided to the culture broth under elevated pressure would be sufficient to triple mcl-PHA productivity in our chemostat system from 3.4 (at 1 bar) to 11 g L(-1)h(-1) (at 3.2 bar).

New insights on the reorganization of gene transcription in Pseudomonas putida KT2440 at elevated pressure

“…We showed previously that the physiology of P. putida KT2440 was hardly affected at 7 bar [2]. No decrease of cell growth rate and PHA-free biomass (total cell dry weight minus PHA weight) production was observed and the respiratory quotient did not vary under these conditions, either.…”

“…Detailed description of these cultivations is reported in a previous work where the effect of pressure on cell physiology was investigated [2]. In the present work we focus on the effect of pressure on the cell transcriptome while using the same biomass samples.…”

“…In particular, this method has proved suitable for the production of medium-chain-length polyhydroxyalkanoates (mcl-PHA), biocompatible and biodegradable polymers with a wide range of applications [11-13], by Pseudomonas putida KT2440. Indeed, we demonstrated in a recent work that except for a small decrease of viability, the physiology of P. putida KT2440 (biomass production, nutrient yields, respiratory quotient, mcl-PHA production) was not impaired at 7 bar [2]. Nevertheless, pressure may still induce stress and affect metabolic pathways, cellular machineries and cellular functions when varying from optimal values, in a similar way as deviations of temperature, pH, osmolarity or water availability.…”

Pilot-scale Production of Functionalized mcl-PHA from Grape Pomace Supplemented with Fatty Acids

“…This ratio is in good agreement with the relative concentrations of C8 and C11:1 (50/50 mol %) fed to the cell culture. Nevertheless, the content of 3-OH-C10 (5.5 mol %) was larger than expected, since P. putida KT2440 is known to synthesize polymer with less than 1 mol % 3-OH-C10 on octanoic acid only [31][32] . The additional 3-OH-C10 monomers most probably arose from the residual amounts of fructose still present after nitrogen limitation that could be metabolized into mcl-PHA via de novo fatty acid synthesis.…”

Strategies for Large-scale Production of Polyhydroxyalkanoates

“…78 The set-up comprised batch cultivation on a C8 carbon source, fed-batch cultivation on C8/C11:1, and finally a continuous cultivation on C8/C11:1 at a dilution rate of 0.15 h -1 . The mcl-PHA content, yield on substrate and volumetric productivity were all found to increase at elevated pressure.…”