Determination of Cell Concentration and Characterization of Cells

Reardon, Kenneth F.; Scheper, Thomas

doi:10.1002/9783527620852.ch6

Cited by 28 publications

(7 citation statements)

References 182 publications

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“…For mycelial fermentations, changes in cell morphology significantly affect broth viscosity . Fermentation broth viscosity is determined by both biomass concentration and liquor properties . Both protein concentration and DNA concentration will significantly influence the broth viscosity if present in high enough quantities.…”

Section: Introductionmentioning

confidence: 99%

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Newton

Schofield

Vlahopoulou³

et al. 2016

Biotechnology Progress

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Monitoring the physical or chemical properties of cell broths to infer cell status is often challenging due to the complex nature of the broth. Key factors indicative of cell status include cell density, cell viability, product leakage, and DNA release to the fermentation broth. The rapid and accurate prediction of cell status for hosts with intracellular protein products can minimise product loss due to leakage at the onset of cell lysis in fermentation. This article reports the rheological examination of an industrially relevant E. coli fermentation producing antibody fragments (Fab'). Viscosity monitoring showed an increase in viscosity during the exponential phase in relation to the cell density increase, a relatively flat profile in the stationary phase, followed by a rapid increase which correlated well with product loss, DNA release and loss of cell viability. This phenomenon was observed over several fermentations that a 25% increase in broth viscosity (using induction‐point viscosity as a reference) indicated 10% product loss. Our results suggest that viscosity can accurately detect cell lysis and product leakage in postinduction cell cultures, and can identify cell lysis earlier than several other common fermentation monitoring techniques. This work demonstrates the utility of rapidly monitoring the physical properties of fermentation broths, and that viscosity monitoring has the potential to be a tool for process development to determine the optimal harvest time and minimise product loss. © 2016 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers, 32:1069–1076, 2016

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Section: Introductionmentioning

confidence: 99%

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Newton

Schofield

Vlahopoulou³

et al. 2016

Biotechnology Progress

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“…For low‐to‐moderate turbidity cultivation broths, probes based on transmission or 90° scattering are best (Fig. ). Reflection measurement is primarily used for high cell densities.…”

Section: Bioprocess Monitoring Systemsmentioning

confidence: 99%

Sensor systems for bioprocess monitoring

Biechele

Busse

Solle

et al. 2015

Engineering in Life Sciences

171

158

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The ability to measure all process variables is of great importance in the field of bioprocess monitoring and control, and continuous, real‐time measurements are highly desired. The more complete and real‐time the measurements are, the more stable, reproducible, and efficient the process can be, leading to reproducibly high‐quality products. This additional information allows the operator to better document the entire process. The process analytical technologies initiative of the US Food and Drug Administration is strongly related to the analysis and control of biopharmaceutical processes. The aim of the initiative is to create processes, generating products of ensured quality by measuring quality‐related process variables. The quality of the product is enhanced by a deep understanding of the process, which is enabled by an effective and suitable sensor system. The aim of this review is to provide an overview of current and emerging sensors for bioprocess monitoring. Sensors directly interfaced to bioreactors for measuring important variables from the gas phase, such as oxygen and carbon dioxide concentration, are discussed, as well as sensors for the monitoring of the biomass concentration and morphology and of the changing medium composition. Furthermore, sensor systems are discussed. These involve sensors (especially biosensors) that are not implemented directly inside the bioreactor but rather are used in conjunction with sample‐taking systems such as flow injection analysis. A major focus is given to spectroscopic sensors, which are noninvasive and offer interesting options for a simultaneous analysis of various compounds. Since data handling is extremely important for this kind of sensor, chemometrics are also included. Soft sensors are discussed as technology that allows a user to incorporate more process data as it become available. Finally, the current state of disposable sensor technology is presented. These sensors are needed for the growing area of disposable and continuous biomanufacturing.

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Section: Common Themes In Bacillus Cereus Research and The Utility Ofmentioning

confidence: 99%

“…As well as measurement of growth rate, FCM has applications including the study during growth and/or differing growth conditions of cell physiology (Cronin and Wilkinson 2008c), changes in cell size (reflected by changes in FSC properties of cells; Robertson and Button 1999), changes in cytoplasmic density (reflected by changes in SSC properties of cells; Davey 1994), DNA (Reardon and Scheper 1993; Stecchini et al. 2001) and lipid or protein contents (Reardon and Scheper 1993) of B. cereus. The physiological status of cells may be assessed by staining with a number of fluorescent dyes to detect alterations in membrane permeability, enzyme activity, redox activity, membrane potential, oxidative damage to DNA and intracellular Ca 2+ and pH (Coder 1997; Nebe‐von‐Caron et al.…”

Section: Common Themes In Bacillus Cereus Research and The Utility Ofmentioning

confidence: 99%

“…Any technique capable of simultaneously measuring up to 12 parameters per cell (such as FCM) is bound to enhance the collection of phenotypic data that can be then used to further characterize and categorize strains. Phenotypic data that may be collected using FCM include enzyme activities, antibiotic resistance, cell size, refractive index, presence of para‐sporal crystals, numbers of cells comprising each CFU and genome size (Reardon and Scheper 1993; Porter et al. 1996; Jacobsen and Jakobsen 1999; Nebe‐von‐Caron et al.…”

Section: Common Themes In Bacillus Cereus Research and The Utility Ofmentioning

confidence: 99%

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The potential of flow cytometry in the study of Bacillus cereus

Cronin

Wilkinson

2010

Journal of Applied Microbiology

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Summary Flow cytometry (FCM) is a rapid method allowing the acquisition of multiparametric data from thousands of individual cells within a sample. As well as measuring the intrinsic light scattering properties of cells, a plethora of fluorescent dyes may be employed to yield information on macromolecule content, surface antigens present or physiological status. Despite FCM’s indispensability within other fields e.g. immunology, it is underutilized within microbiological research. In this review, a strong case is presented for the potential of FCM in the study of Gram‐positive spore‐former, Bacillus cereus. Previous reports where FCM was successfully used in the study of B. cereus are reviewed along with relevant studies involving other members of the genus. Under headings reflecting common research themes associated with B. cereus, specific instances where FCM has generated novel data, providing a unique insight into the organism, are discussed. Further applications are posited, based on the authors’ own research with FCM and B. cereus and work extant in the broader field of microbial cytometry. The authors conclude that, while the expense of equipment and reagents is an undeniable disadvantage, FCM is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique.

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Determination of Cell Concentration and Characterization of Cells

Cited by 28 publications

References 182 publications

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Detecting cell lysis using viscosity monitoring in E. coli fermentation to prevent product loss

Sensor systems for bioprocess monitoring

The potential of flow cytometry in the study of Bacillus cereus

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