Differences in growth physiology and aggregation of <i>Pichia pastoris</i> cells between solid‐state and submerged fermentations under aerobic conditions

Lima-Pérez, Jorge; Rodriguez-Gomez, Divanery; Loera, Octavio; Viniegra‐González, Gustavo; López-Pérez, Marcos

doi:10.1002/jctb.5384

Cited by 12 publications

(1 citation statement)

References 16 publications

(23 reference statements)

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“…The first limitation is the aggregation behavior of P. pastoris cells, as shown by our microscopic visualization and observed in a previous study. 71 Therefore, the daughter cell content determined by the Gaussian mixture analysis would be higher if P. pastoris cells were present in the single-cell stage. This limitation can be overcome by optimizing the staining method; for example, by changing the buffer system or pH.…”

Section: Discussionmentioning

confidence: 99%

Autofluorescence prediction model for fluorescence unmixing and age determination

Eigenfeld

Kerpes

Whitehead

et al. 2022

Biotechnology Journal

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Background: Flow cytometry is a powerful tool for identifying and quantifying various cell markers, such as viability, vitality, and individual cell age, at single-cell stages. However, cell autofluorescence and marker fluorophore signals overlap at low fluorescence intensities. Thus, these signals must be unmixed before determining the age fraction. Methods and Results:A comparison was made between principal component regression (PCR) and random forest (RF) to predict autofluorescence signals of Saccharomyces pastorianus var. carlsbergensis in a flow cytometer. RF provided better prediction results than the PCR and was therefore determined to be better suited for unmixing signals. In the subsequent application for unmixing the autofluorescence signal from the marker fluorophore signal, the Gaussian mixture analysis based on RF was in better agreement with the microscopy-determined replicative age distribution than the PCR-based method. Conclusion:The proposed approach of single-laser spectral unmixing and subsequent Gaussian mixture analysis showed that the microscopy data was consistent with the unmixed fluorescence spectra. The demonstrated approach enables fast and reliable unmixing of flow cytometric spectral data using a single-laser spectral unmixing method. This analysis method enables age determination of cells in industrial processes. This age determination allows for quantifying the yeast cell's age fractions, providing a detailed view of age-related changes. Additionally, the bud scar labeling technique can be used to determine age-related changes in Pichia pastoris yeast for biotechnological applications or recombinant protein expression.

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Section: Discussionmentioning

confidence: 99%

Autofluorescence prediction model for fluorescence unmixing and age determination

Eigenfeld

Kerpes

Whitehead

et al. 2022

Biotechnology Journal

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Differences in the physiology of Pichia pastoris grown on glycerol/glucose using polyurethane foam slabs as solid support

Zárate‐Bonilla,

Rodríguez‐Gómez,

Viniegra‐González

et al. 2024

J of Chemical Tech & Biotech

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BACKGROUNDGrowth physiology and metabolic flux of Pichia pastoris were analyzed using glass columns containing slabs of polyurethane foam as a solid support and culture medium with glycerol or glucose as a carbon source.RESULTSMedia with glycerol produced an average of 30.91% more biomass than media where glucose was used. However, the specific growth rate registered higher values for media with glucose than those with glycerol at low substrate concentrations: 0.46 versus 0.22 h−1. Media with glycerol produced a large amount of d‐arabitol, whereas ethanol and citrate were the predominant metabolites in media with glucose. Both types of culture systems generated biphasic curves in terms of the respiratory quotient value, whose inflection point coincided with the transition from aerobic metabolism to a mixed one.CONCLUSIONAdequate oxygen transfer occurred in both culture systems; therefore, the differences found in growth physiology can be explained by the transport mechanism that internalizes glycerol, the access point to primary metabolism, and the degree of glycerol oxidation. The arrangement of cells in the polyurethane matrix allowed an attenuation of the repression phenomenon due to excess substrate for both carbon sources. This is the first work to study metabolic flux comparisons using this system. © 2024 Society of Chemical Industry (SCI).

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Solid-state fermentation of Bacillus thuringiensis var kurstaki HD-73 maintains higher biomass and spore yields as compared to submerged fermentation using the same media

Lima-Pérez

López-Pérez

Viniegra‐González

et al. 2019

Bioprocess Biosyst Eng

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Differences in growth physiology and aggregation of Pichia pastoris cells between solid‐state and submerged fermentations under aerobic conditions

Cited by 12 publications

References 16 publications

Autofluorescence prediction model for fluorescence unmixing and age determination

Autofluorescence prediction model for fluorescence unmixing and age determination

Differences in the physiology of Pichia pastoris grown on glycerol/glucose using polyurethane foam slabs as solid support

Solid-state fermentation of Bacillus thuringiensis var kurstaki HD-73 maintains higher biomass and spore yields as compared to submerged fermentation using the same media

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