Studying the impact of cell age on the yeast growth behaviour of<i>Saccharomyces pastorianus</i>var.<i>carlsbergensis</i>by magnetic separation

Eigenfeld, Marco; Wittmann, Leonie; Kerpes, Roland; Schwaminger, Sebastian P.; Becker, Thomas

doi:10.1002/biot.202200610

Cited by 6 publications

(17 citation statements)

References 68 publications

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“…These results highlight for the first time the potential of using magnetic nanoparticles in yeast cell fermentation without affecting the cells' viability, thus enabling further applications that combine the metabolic activity of yeast cells with the magnetic properties of nanoparticles [76], e.g. magnetic cell separation for yeast repitching.…”

Section: Discussionmentioning

confidence: 80%

Quantification methods of determining brewer’s and pharmaceutical yeast cell viability: accuracy and impact of nanoparticles

et al. 2023

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For industrial processes, a fast, precise, and reliable method of determining the physiological state of yeast cells, especially viability, is essential. However, an increasing number of processes use magnetic nanoparticles (MNPs) for yeast cell manipulation, but their impact on yeast cell viability and the assay itself is unclear. This study tested the viability of Saccharomyces pastorianus ssp. carlsbergensis and Pichia pastoris by comparing traditional colourimetric, high-throughput, and growth assays with membrane fluidity. Results showed that methylene blue staining is only reliable for S. pastorianus cells with good viability, being erroneous in low viability (R2 = 0.945; $$\widehat{\sigma }$$ σ ^ = 5.78%). In comparison, the fluorescence microscopy–based assay of S. pastorianus demonstrated a coefficient of determination of R2 = 0.991 at $$\alpha =0$$ α = 0 ($$\widehat{\sigma }$$ σ ^ = 2.50%) and flow cytometric viability determination using carboxyfluorescein diacetate (CFDA), enabling high-throughput analysis of representative cell numbers; R2 = 0.972 ($$\alpha =0$$ α = 0 ; $$\widehat{\sigma }$$ σ ^ = 3.89%). Membrane fluidity resulted in a non-linear relationship with the viability of the yeast cells ($$\alpha \ne 0$$ α ≠ 0 ). We also determined similar results using P. pastoris yeast. In addition, we demonstrated that MNPs affected methylene blue staining by overestimating viability. The random forest model has been shown to be a precise method for classifying nanoparticles and yeast cells and viability differentiation in flow cytometry by using CFDA. Moreover, CFDA and membrane fluidity revealed precise results for both yeasts, also in the presence of nanoparticles, enabling fast and reliable determination of viability in many experiments using MNPs for yeast cell manipulation or separation.

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Section: Discussionmentioning

confidence: 80%

Quantification methods of determining brewer’s and pharmaceutical yeast cell viability: accuracy and impact of nanoparticles

et al. 2023

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“…As previously described, the MNPs were further functionalized with EDTA by an amide bonding. 25 Briefly, 179 mg of iron oxide nanoparticles were suspended in 100 mL of Millipore water, ultrasonicated on ice (3 min, 20%, 10 sec on, 15 sec off, 20 kHz, Branson Ultrasonics), mixed with citric acid (Carl Roth, c = 0.029 mol L −1 , V = 100 mL, ultrasonicated), and ultrasonicated as before. After 15 min incubation, the pH was adjusted to 11 using tetramethylammonium hydroxide (Sigma Aldrich).…”

Section: A Synthesis Of Ethylenediaminetetraacetic Acid-functionalize...mentioning

confidence: 99%

“…The specificity of the nanoparticle binding has been verified before. 25 Microscopic images are taken by a Zeiss Axio Observer 7, 100× objective using bright field and fluorescent channels. The extinction wavelength was 480 nm and the emission wavelength was 505 nm.…”

Section: B Yeast Cell Labelingmentioning

confidence: 99%

“…19,22,23 The hydrodynamic drag force originates from the Stokes equations, with particles in laminar flow aligning with fluid streamlines based on size due to the inertial force. 24 Integrating size separation with magnetic bud scar labeling 25,26 offers a promising method for age-based yeast cell fractionation (Fig. 1).…”

Section: A Introductionmentioning

confidence: 99%

“…2a). 25,26 In designing this millifluidic magnetophoretic process, several critical factors must be considered:…”

Section: A Introductionmentioning

confidence: 99%

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Millifluidic magnetophoresis-based chip for age-specific fractionation: evaluating the impact of age on metabolomics and gene expression in yeast

Wittmann,

Eigenfeld,

Büchner

et al. 2024

Lab Chip

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Transcriptional Downregulation of Methanol Metabolism Key Genes During Yeast Death in Engineered Pichia pastoris

Wang,

Jiang,

et al. 2024

Biotechnology Journal

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Pichia pastoris possesses the unique ability to utilize methanol as its sole carbon source, which makes it a proper host for producing various high‐value‐added products via metabolic engineering. Nevertheless, cell death has been observed during the fermentation of modified P. pastoris, with limited literature elucidating the underlying causes and mechanisms. Understanding the death mechanisms during methanol‐based fermentation is crucial for optimizing fermentation strategies, enhancing the accumulation of target products, and reducing production costs. Here, we first sought to eliminate the potential causes of cell death during fermentation, such as inadequate inorganic salts and toxic by‐product accumulation. The elimination of these potential causes was achieved efficiently utilizing the high‐throughput fermentation equipment. Subsequently, we established a correlation between yeast cell death and the duration of the methanol metabolism period by monitoring the growth of the yeast at different fermentation stages. A critical revelation from this work came from analyzing the yeast's transcriptomic data at various stages of methanol metabolism. It was observed that a significant characteristic of yeast cell death during fermentation was the marked down‐regulation of transcript levels of key enzymes involved in the methanol assimilation pathway and genes related to their biosynthesis process. The findings of this work are crucial for better understanding the causes and mechanisms of cell death for engineered P. pastoris during methanol‐utilized fermentation.

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Studying the impact of cell age on the yeast growth behaviour ofSaccharomyces pastorianusvar.carlsbergensisby magnetic separation

Cited by 6 publications

References 68 publications

Quantification methods of determining brewer’s and pharmaceutical yeast cell viability: accuracy and impact of nanoparticles

Quantification methods of determining brewer’s and pharmaceutical yeast cell viability: accuracy and impact of nanoparticles

Millifluidic magnetophoresis-based chip for age-specific fractionation: evaluating the impact of age on metabolomics and gene expression in yeast

Transcriptional Downregulation of Methanol Metabolism Key Genes During Yeast Death in Engineered Pichia pastoris

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