Dielectric monitoring of growth and sporulation of Bacillus thuringiensis

Sarrafzadeh, Mohammad‐Hossein; Belloy, L.; Esteban, Geoffrey; Navarro, J. M.; Ghommidh, C.

doi:10.1007/s10529-005-2543-x

Cited by 56 publications

(26 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Permittivity is a direct measure of the membrane enclosed volume fraction or biovolume of the cell suspension. These measurements also reflect changes in cell physiology which has been demonstrated for several cell types (Fehrenbach et al 1992;Noll and Biselli 1998;Zeiser et al 2000;Ducommun et al 2002;Cannizzaro et al 2003;Sarrafzadeh et al 2005).…”

mentioning

confidence: 61%

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

2010

View full text Add to dashboard Cite

Changes in the nutrient availability of mammalian cell cultures are reflected in the bdispersion parameter characteristic frequency (f C ) and the on-line dual frequency permittivity signal. Multifrequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f C occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f C indicated nutrient limitations. f C correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f C . When significant cell size changes occurred during the cultivations, the analysis of the b-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.

show abstract

mentioning

confidence: 61%

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

2010

View full text Add to dashboard Cite

show abstract

“…For the first protocol, the optical density at 600 nm (OD 600 ) of the culture was monitored at 24, 48, 72, and 96 h, a measurement that reflects the density of the particles in suspension. Since spores are less dense than vegetative cells, the OD 600 should decrease as the bacilli in the culture become spores (20,21). For the second technique, samples were obtained at the time points listed above and assessed by phase microscopy for sporulation.…”

Section: Methodsmentioning

confidence: 99%

Bacillus anthracis Exosporium Protein BclA Affects Spore Germination, Interaction with Extracellular Matrix Proteins, and Hydrophobicity

et al. 2007

View full text Add to dashboard Cite

Bacillus collagen-like protein of anthracis (BclA) is the immunodominant glycoprotein on the exosporium of Bacillus anthracis spores. Here, we sought to assess the impact of BclA on spore germination in vitro and in vivo, surface charge, and interaction with host matrix proteins. For that purpose, we constructed a markerless bclA null mutant in B. anthracis Sterne strain 34F2. The growth and sporulation rates of the ⌬bclA and parent strains were nearly indistinguishable, but germination of mutant spores occurred more rapidly than that of wild-type spores in vitro and was more complete by 60 min. Additionally, the mean time to death of A/J mice inoculated subcutaneously or intranasally with mutant spores was lower than that for the wild-type spores even though the 50% lethal doses of the two strains were similar. We speculated that these in vitro and in vivo differences between mutant and wild-type spores might reflect the ease of access of germinants to their receptors in the absence of BclA. We also compared the hydrophobic and adhesive properties of ⌬bclA and wild-type spores. The ⌬bclA spores were markedly less water repellent than wild-type spores, and, probably as a consequence, the extracellular matrix proteins laminin and fibronectin bound significantly better to mutant than to wild-type spores. These studies suggest that BclA acts as a shield to not only reduce the ease with which spores germinate but also change the surface properties of the spore, which, in turn, may impede the interaction of the spore with host matrix substances.Bacillus anthracis is a gram-positive, spore-forming bacillus that can cause anthrax (15). The spore is the form of the organism found in its natural habitat, the soil, and is also the infectious form for herbivores, the typical vertebrate host for the bacterium, and humans (15). The B. anthracis spore is covered by a loose balloon-like membranous structure called the exosporium (8). BclA (for bacillus collagen-like protein of anthracis) was first described by Sylvestre et al. (23), who constructed an insertional bclA mutant and compared it to its wild-type parent. These investigators and, subsequently, others found that BclA is a glycoprotein and a major component of the hair-like projections that cover the exosporium (16,22,23,25). BclA is also an immunodominant marker on the outside of the spore (22). The finding that BclA does not play a significant role in the virulence of a Sterne-like strain for mice was first reported by Sylvestre et al. (23). Sterne strains contain pXO1 but not pX02 and are attenuated in humans and many other animals except certain mouse strains (26). In support of the findings of Sylvestre and colleagues, Bozue and coworkers recently constructed a bclA mutant of the fully virulent B. anthracis Ames strain and showed that the absence of BclA had no impact on the lethality of that strain for guinea pigs or mice (5). Whether BclA, the substance on the spore with which the host cells probably first interact, plays a more subtle role in B. anthracis patho...

show abstract

“…[18][19][20][21][22][23][24] Permittivity has been particularly advantageous in monitoring the biomass of cultures that are otherwise difficult to characterize, such as those with high amounts of insoluble matter, 18 heavily flocculating cultures, 25 and spore-forming cultures. 26 In mammalian cultures, permittivity has been used for biomass monitoring, 27,28 specific rate calculations, 29 and bioreactor control. 30 Guan et al 31,32 used dielectric spectroscopy along with heat flow rate measurements to relate specific heat flow with various metabolic rate parameters, and then applied those parameters as a basis for medium optimization.…”

Section: Eðf þ ¼mentioning

confidence: 99%

Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed‐batch CHO processes using dielectric spectroscopy

Opel

Amanullah

2010

Biotechnology Progress

View full text Add to dashboard Cite

Dielectric spectroscopy was used to analyze typical batch and fed-batch CHO cell culture processes. Three methods of analysis (linear modeling, Cole-Cole modeling, and partial least squares regression), were used to correlate the spectroscopic data with routine biomass measurements [viable packed cell volume, viable cell concentration (VCC), cell size, and oxygen uptake rate (OUR)]. All three models predicted offline biomass measurements accurately during the growth phase of the cultures. However, during the stationary and decline phases of the cultures, the models decreased in accuracy to varying degrees. Offline cell radius measurements were unsuccessfully used to correct for the deviations from the linear model, indicating that physiological changes affecting permittivity were occurring. The beta-dispersion was analyzed using the Cole-Cole distribution parameters Deltaepsilon (magnitude of the permittivity drop), f(c) (critical frequency), and alpha (Cole-Cole parameter). Furthermore, the dielectric parameters static internal conductivity (sigma(i)) and membrane capacitance per area (C(m)) were calculated for the cultures. Finally, the relationship between permittivity, OUR, and VCC was examined, demonstrating how the definition of viability is critical when analyzing biomass online. The results indicate that the common assumptions of constant size and dielectric properties used in dielectric analysis are not always valid during later phases of cell culture processes. The findings also demonstrate that dielectric spectroscopy, while not a substitute for VCC, is a complementary measurement of viable biomass, providing useful auxiliary information about the physiological state of a culture.

show abstract

Dielectric monitoring of growth and sporulation of Bacillus thuringiensis

Cited by 56 publications

References 6 publications

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

Bacillus anthracis Exosporium Protein BclA Affects Spore Germination, Interaction with Extracellular Matrix Proteins, and Hydrophobicity

Quantitative modeling of viable cell density, cell size, intracellular conductivity, and membrane capacitance in batch and fed‐batch CHO processes using dielectric spectroscopy

Contact Info

Product

Resources

About