Rheological Behavior of High Cell Density Pseudomonas putida LS46 Cultures during Production of Medium Chain Length Polyhydroxyalkanoate (PHA) Polymers

The rheology of high-cell density (HCD) cultures is an important parameter for its impact on mixing and sparging, process scale-up, and downstream unit operations in bioprocess development. In this work, time-dependent rheological properties of HCD Pseudomonas putida LS46 cultures were monitored for microbial polyhydroxyalkanoate (PHA) production. As the cell density of the fed-batch cultivation increased (0 to 25 g·L−1 cell dry mass, CDM), the apparent viscosity increased nearly nine-fold throughout the fed-batch process. The medium behaved as a nearly Newtonian fluid at lower cell densities, and became increasingly shear-thinning as the cell density increased. However, shear-thickening behavior was observed at shearing rates of approximately 75 rad·s−1 or higher, and its onset increased with viscosity of the sample. The supernatant, which contained up to 9 g·L−1 soluble organic material, contributed more to the observed viscosity effect than did the presence of cells. Owing to this behavior, the oxygen transfer performance of the bioreactor, for otherwise constant operating conditions, was reduced by 50% over the cultivation time. This study has shown that the dynamic rheology of HCD cultures is an important engineering parameter that may impact the final outcome in PHA cultivations. Understanding and anticipating this behavior and its biochemical origins could be important for improving overall productivity, yield, process scalability, and the efficacy of downstream processing unit operations.

Download Full-text

Poly(3-Hydroxybutyrate) Production from Glycerol by Zobellella denitrificans MW1 via High-Cell-Density Fed-Batch Fermentation and Simplified Solvent Extraction

Applied and Environmental Microbiology ◽

10.1128/aem.01162-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6222-6231 ◽

Cited By ~ 92

Author(s):

Mohammad H. A. Ibrahim ◽

Alexander Steinbüchel

Keyword(s):

Solvent Extraction ◽

Cell Density ◽

High Cell Density ◽

Scale Up ◽

Downstream Processing ◽

Production Costs ◽

Yield Coefficient ◽

High Cell ◽

Fed Batch ◽

High Specific Growth Rate

ABSTRACT Industrial production of biodegradable polyesters such as polyhydroxyalkanoates is hampered by high production costs, among which the costs for substrates and for downstream processing represent the main obstacles. Inexpensive fermentable raw materials such as crude glycerol, an abundant by-product of the biodiesel industry, have emerged to be promising carbon sources for industrial fermentations. In this study, Zobellella denitrificans MW1, a recently isolated bacterium, was used for the production of poly(3-hydroxybutyrate) (PHB) from glycerol as the sole carbon source. Pilot-scale fermentations (42-liter scale) were conducted to scale up the high PHB accumulation capability of this strain. By fed-batch cultivation, at first a relatively high cell density (29.9 ± 1.3 g/liter) was obtained during only a short fermentation period (24 h). However, the PHB content was relatively low (31.0% ± 4.2% [wt/wt]). Afterwards, much higher concentrations of PHB (up to 54.3 ± 7.9 g/liter) and higher cell densities (up to 81.2 ± 2.5 g/liter) were obtained by further fed-batch optimization in the presence of 20 g/liter NaCl, with optimized feeding of glycerol and ammonia to support both cell growth and polymer accumulation over a period of 50 h. A high specific growth rate (0.422/h) and a short doubling time (1.64 h) were attained. The maximum PHB content obtained was 66.9% ± 7.6% of cell dry weight, and the maximum polymer productivity and substrate yield coefficient were 1.09 ± 0.16 g/liter/h and 0.25 ± 0.04 g PHB/g glycerol, respectively. Furthermore, a simple organic solvent extraction process was employed for PHB recovery during downstream processing: self-flotation of cell debris after extraction of PHB with chloroform allowed a convenient separation of a clear PHB-solvent solution from the cells. Maximum PHB recovery (85.0% ± 0.10% [wt/wt]) was reached after 72 h of extraction with chloroform at 30°C, with a polymer purity of 98.3% ± 1.3%.

Download Full-text

Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440

Applied Microbiology and Biotechnology ◽

10.1007/s00253-005-0191-7 ◽

2006 ◽

Vol 71 (4) ◽

pp. 423-431 ◽

Cited By ~ 52

Author(s):

Zhiyong Sun ◽

Juliana A. Ramsay ◽

Martin Guay ◽

Bruce A. Ramsay

Keyword(s):

Pseudomonas Putida ◽

Cell Density ◽

High Cell Density ◽

Batch Cultivation ◽

Feeding Strategies ◽

High Cell ◽

Fed Batch ◽

Pseudomonas Putida Kt2440 ◽

Fed Batch Cultivation

Download Full-text

Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c4 by high-cell-density fed-batch cultivation of Pseudomonas putida

Protein Expression and Purification ◽

10.1016/s1046-5928(02)00575-2 ◽

2003 ◽

Vol 27 (1) ◽

pp. 175-181 ◽

Cited By ~ 9

Author(s):

Marianne H Thuesen ◽

Allan Nørgaard ◽

Anne M Hansen ◽

Mikael B Caspersen ◽

Hans E.M Christensen

Keyword(s):

Pseudomonas Putida ◽

Cell Density ◽

High Cell Density ◽

Pseudomonas Stutzeri ◽

Batch Cultivation ◽

High Cell ◽

Fed Batch ◽

Fed Batch Cultivation

Download Full-text

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

Bioengineering ◽

10.3390/bioengineering6040089 ◽

2019 ◽

Vol 6 (4) ◽

pp. 89 ◽

Cited By ~ 5

Author(s):

Warren Blunt ◽

Christopher Dartiailh ◽

Richard Sparling ◽

Daniel J. Gapes ◽

David B. Levin ◽

...

Keyword(s):

Pseudomonas Putida ◽

Cell Density ◽

Chain Length ◽

Octanoic Acid ◽

High Cell Density ◽

High Cell ◽

Fed Batch ◽

Medium Chain Length ◽

Pulse Feed ◽

Feed Strategy

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.

Download Full-text