High cell density cultivation of Pseudomonas putida KT2440 using glucose without the need for oxygen enriched air supply (original) (raw)
Related papers
Applied Microbiology and Biotechnology, 2006
Four automatic substrate feeding strategies were developed and investigated in this study to obtain rapid, repeatable, and reliable high cell densities of Pseudomonas putida KT2440 from glucose. Growth yield data of the key nutrients, Y X/Glucose , Y X/NH4 , Y X/PO4 , Y X/Mg , and Y CO2/Glucose , were determined to be 0.41, 5.44, 13.70, 236, and 0.65 g g −1 , respectively. Although standard exponential feeding strategy worked well when the predetermined μ was set at 0.25 h −1 , an exponential glucose feeding strategy with online μ max estimation resulted in a higher average biomass productivity (3.4 vs 2.8 g l −1 h −1 ). A CO 2 production rate based pulse glucose feeding strategy also resulted in good overall productivity (3.0 g l −1 h −1 ) and can be used as an alternative to pH-stat or DO-stat feeding. A cumulative CO 2 production based continuous feed with real-time cumulative glucose consumption estimation resulted in much higher biomass productivity (4.3 g l −1 h −1 ) and appears to be an excellent and reliable approach to fully automating high-cell-density fed-batch cultivation of P. putida.
Applied microbiology and biotechnology, 2014
A mathematically based fed-batch bioprocess demonstrated the suitability of using a relatively cheap and renewable substrate (butyric acid) for Pseudomonas putida CA-3 high cell density cultivation. Butyric acid fine-tuned addition is critical to extend the fermentation run and avoid oxygen consumption while maximising the biomass volumetric productivity. A conservative submaximal growth rate (μ of 0.25 h(-1)) achieved 71.3 g L(-1) of biomass after 42 h of fed-batch growth. When a more ambitious feed rate was supplied in order to match a μ of 0.35 h(-1), the volumetric productivity was increased to 2.0 g L(-1) h(-1), corresponding to a run of 25 h and 50 g L(-1) of biomass. Both results represent the highest biomass and the best biomass volumetric productivity with butyrate as a sole carbon source. However, medium chain length polyhydroxyalkanoate (mcl-PHA) accumulation with butyrate grown cells is low (4 %). To achieve a higher mcl-PHA volumetric productivity, decanoate was supplie...
Applied Biochemistry and Biotechnology, 2004
We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA MCL ) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65 h -1 . Limitation and inhibition owing to NH 4 + ions were observed, respectively, at 400 and 3500 mg of NH 4 + /L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA MCL . Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA MCL content. Using the last strategy, 21% PHA MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH 4 + limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h 52 Diniz et al.
Applied biochemistry and biotechnology, 2004
We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA MCL ) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65 h -1 . Limitation and inhibition owing to NH 4 + ions were observed, respectively, at 400 and 3500 mg of NH 4 + /L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA MCL . Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA MCL content. Using the last strategy, 21% PHA MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH 4 + limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h 52 Diniz et al.
Bioengineering, 2019
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 vo...
Biotechnology progress, 2018
The growth rate of four strains of Pseudomonas putida, KT2440, KT2442, KTH2 and KTH2 (pESOX3), under different fluid dynamic conditions has been studied. The cultures were conducted in a stirred tank bioreactor by changing the stirrer speed. Several process variables, such as biomass concentration, dissolved oxygen concentration, oxygen mass transfer rate and oxygen uptake rate, have been measured or calculated. Also cell viability was determined by viable colony counting in Petri dishes and culture samples were subjected into a transmission electron microscopy analysis, in order to describe the integrity of the individual cells. The experimental results show that the genetically modified organisms, the strains KTH2 and KTH2 (pESOX3), present a different growth under low agitation conditions, and low oxygen supply level, while the growth of the wild type strains, KT2440 and KT2442, followed the typical sigmoidal evolution that could be described by the logistic equation. The presenc...
Journal of Biotechnology, 2007
A method was developed to increase the yield of MCL-PHA from nonanoic acid in the PHA accumulation phase. Pseudomonas putida KT2440 was grown on glucose until ammonium-limitation was imposed. In the second (accumulation) stage, either glucose, nonanoic acid, or a mixture of these carbon and energy sources was supplied. Since the medium-chain-length poly-3-hydroxyalkanoate (MCL-PHA) subunits produced are unique for each carbon source, their relative contribution to PHA yield could be calculated. Y (C7+C9)/NA was 0.254 mol mol −1 during PHA synthesis from nonanoic acid. Y (C8+C10)/G was only 0.057 mol mol −1 during PHA synthesis from glucose. When nonanoic acid and glucose were fed together, Y (C7+C8)/NA almost doubled to 0.450 mol mol −1 while Y (C8+C10)/G decreased to 0.011 mol mol −1. These results demonstrate that substantial savings can be obtained by feeding glucose with substrates that are good for PHA production but much more expensive than glucose.
Flux balance analysis for maximizing polyhydroxyalkanoate production in Pseudomonas putida
The aim of this work is to develop process strategies to understand the behaviour of Psendomonas putida for cost-effective synthesis of mcl-PHA with the help of metabolic flux model developed using linear programming. After screening and selection from various carbon sources in shake flask experiments, 2 carbon sources, namely, glucose and glycerol were selected for further studies. The possibilities for higher PHA biosynthesis were illustrated by the flux balance analysis (FBA) supplemented with continuous cultivation data. The optimal ratio between glucose, glycerol and nitrogen is obtained by simulation of the metabolic model. But the cost of raw materials is the tailback for the cost-effective production of mcl-PHA. Hence, a metabolic flux distribution model was developed for dual substrate utilization by co-feeding glucose and glycerol for optimal PHA synthesis. Response surface results indicated that the highest PHA accumulation conditions for glucose and glycerol.
Microbial Cell Factories, 2011
Background: The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicinresistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.