Effective Dissolved Oxygen Control Strategy for High-Cell-Density Cultures

Cárcamo, Martín; Saa, Pedro A.; Torres, Jorge; Torres, Samuel; Mandujano, Patricio; Correa, Jose Ricardo Perez; Agosín, Eduardo

doi:10.1109/tla.2014.6827863

Cited by 20 publications

(10 citation statements)

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“…Dissolved Oxygen was maintained above 40% saturation during all the cultivation period. Aerobiosis was achieved by a triple split-range control action, including agitation (200–800 [RPM]), air flow (0.25–1.0 [L/min]) and pure oxygen flow (0–1.0 [L/min]) [ 64 ]. pH was controlled using phosphoric acid 20% [v/v] and sodium hydroxide 20% [v/v].…”

Section: Methodsmentioning

confidence: 99%

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

2017

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Background Pichia pastoris shows physiological advantages in producing recombinant proteins, compared to other commonly used cell factories. This yeast is mostly grown in dynamic cultivation systems, where the cell’s environment is continuously changing and many variables influence process productivity. In this context, a model capable of explaining and predicting cell behavior for the rational design of bioprocesses is highly desirable. Currently, there are five genome-scale metabolic reconstructions of P. pastoris which have been used to predict extracellular cell behavior in stationary conditions.ResultsIn this work, we assembled a dynamic genome-scale metabolic model for glucose-limited, aerobic cultivations of Pichia pastoris. Starting from an initial model structure for batch and fed-batch cultures, we performed pre/post regression diagnostics to ensure that model parameters were identifiable, significant and sensitive. Once identified, the non-relevant ones were iteratively fixed until a priori robust modeling structures were found for each type of cultivation. Next, the robustness of these reduced structures was confirmed by calibrating the model with new datasets, where no sensitivity, identifiability or significance problems appeared in their parameters. Afterwards, the model was validated for the prediction of batch and fed-batch dynamics in the studied conditions.Lastly, the model was employed as a case study to analyze the metabolic flux distribution of a fed-batch culture and to unravel genetic and process engineering strategies to improve the production of recombinant Human Serum Albumin (HSA). Simulation of single knock-outs indicated that deviation of carbon towards cysteine and tryptophan formation improves HSA production. The deletion of methylene tetrahydrofolate dehydrogenase could increase the HSA volumetric productivity by 630%. Moreover, given specific bioprocess limitations and strain characteristics, the model suggests that implementation of a decreasing specific growth rate during the feed phase of a fed-batch culture results in a 25% increase of the volumetric productivity of the protein.ConclusionIn this work, we formulated a dynamic genome scale metabolic model of Pichia pastoris that yields realistic metabolic flux distributions throughout dynamic cultivations. The model can be calibrated with experimental data to rationally propose genetic and process engineering strategies to improve the performance of a P. pastoris strain of interest.Electronic supplementary materialThe online version of this article (doi:10.1186/s12918-017-0408-2) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

2017

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“…H. mediterranei grown on ammonium and nitrate cultures generated 4.6 wt% PHBV with 16.9 mol% 3HV, and 9.3 wt% PHBV containing 12.5 mol% 3HV, respectively . DO levels critically affect cell growth . Halomonas campasalis reached a maximum PHA content of 56.23% under low DO, demonstrating a strong oxygen demand in a growth phase and improved PHA production under low DO .…”

Section: Engineering Halophilic Chassis For Pha Bioproductionmentioning

confidence: 99%

“…DO levels critically affect cell growth . Halomonas campasalis reached a maximum PHA content of 56.23% under low DO, demonstrating a strong oxygen demand in a growth phase and improved PHA production under low DO . When promoter P vgb of a bacterial hemoglobin gene vgb was engineered cascade linked in H. bluephagenesis , better cell growth and enhanced PHB accumulation was observed …”

Section: Engineering Halophilic Chassis For Pha Bioproductionmentioning

confidence: 99%

Halophiles as Chassis for Bioproduction

2018

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Industrial biotechnology aims to solve the challenges of petroleum‐based production using microorganisms as catalysts. However, current industrial biotechnology suffers from high energy and fresh water consumption as well as difficult downstream purification. In addition, most industrial microorganisms are not resistant to other microbial contaminations, and this seriously compromises process effectiveness and the economy. The recently proposed “next‐generation industrial biotechnology” employs contamination resistant microorganisms, including alkaliphilic, acidophilic, thermophilic or halophilic bacteria, etc., for bioproduction. The most successful example is the use of halophilic bacteria as chassis for the production of chemicals, proteins, and biopolymers. This review summarizes some of the most recent studies to engineer the halophilic chassis Halomonas spp. for the production of chemicals, proteins, and several biopolyesters such as poly(3‐hydroxybutyrate), copolymers of 3‐hydroxybutyrate and 3‐hydroxyvalerate, and copolymers of 3‐hydroxybutyrate and 4‐hydroxybutyrate. The Halomonas chassis is also successfully engineered to change its morphology from short bars to long fibers and large spheres for convenient gravity separation. This simplifies the bioprocessing.

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“…The k L a values estimated are within the range of values found in the literature for laboratory scale CSTR bioreactors with a single diffuser. 7,9,11,22,23 Nevertheless, there are several new methodologies to enhance k L a, such as different impeller configurations, which are not included in our bioreactor yet. 9,23 Hence, some k L a values recently reported in the literature are higher than ours.…”

Section: Sensitivity Analysis Using Different Curvesmentioning

confidence: 99%

Automated algorithm to determinek_Laconsidering system delay

Torres

Cerri

Ribeiro

et al. 2017

J. Chem. Technol. Biotechnol

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BACKGROUND Quantification of the volumetric oxygen transfer coefficient (kLa) is essential to characterize and optimize the oxygen transfer capacity of bioreactors. First order methodologies are commonly used to estimate kLa; however, when the delay of the system cannot be neglected, second‐order methodologies are required for accurate estimations. Second‐order methods are time consuming and hard to reproduce. In this study, we describe an automated algorithm to estimate kLa in conventional bioreactors. RESULTS The simple, four step algorithm developed considers: (1) data smoothing; (2) selection of a high oxygen variation zone; (3) selection of the time period where the instantaneous kLa is constant; and (4) kLa estimation. The algorithm was coded in MATLAB and four adjustable parameters were fixed heuristically using eight response curves with different hydrodynamic conditions (varying viscosity, agitation and aeration). Compared with manual processing, 80 validation experiments showed that the proposed automatic algorithm yields much more reproducible results in a fraction of the manual processing time. CONCLUSION The algorithm is fast and yields, without human intervention, reliable kLa estimations under different hydrodynamic conditions; hence, it is useful for designing high throughput kLa assessment systems to optimize oxygen delivery in bioreactors. © 2016 Society of Chemical Industry

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Effective Dissolved Oxygen Control Strategy for High-Cell-Density Cultures

Cited by 20 publications

References 0 publications

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

Halophiles as Chassis for Bioproduction

Automated algorithm to determinek_Laconsidering system delay

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Effective Dissolved Oxygen Control Strategy for High-Cell-Density Cultures

Cited by 20 publications

References 0 publications

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

Dynamic genome-scale metabolic modeling of the yeast Pichia pastoris

Halophiles as Chassis for Bioproduction

Automated algorithm to determinekLaconsidering system delay

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Automated algorithm to determinek_Laconsidering system delay