scholarly journals Effect of different aeration and polyethylene glycol concentration on growth of Escherichia coli DH5α in a 1L bioreactor

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Polyethylene Glycol ◽  
Cell Growth ◽  
Growth Medium ◽  
Growth Rates ◽  
Shake Flask ◽  
Foam Formation ◽  
Biomass Formation ◽  
E Coli ◽  
Toxicity Effect

Large culture volume in bioreactor necessitates aeration for providing sufficient oxygen for cell growth. Thus, extend of aeration and amount of anti-foam needed for suppressing foam formation are key parameters determining the success of bioreactor fermentation. Specifically, while aeration provides more oxygen for powering cellular metabolism and could lead to faster growth rate and more efficient metabolism, it also introduces greater shear stress, mixing and foam formation. On the other hand, anti-foaming agents such as polyethylene glycol (PEG) could exert a toxicity effect on cells as well as introducing increased osmolarity and viscosity that could hamper cell growth. In this preliminary study, effect of different PEG concentrations and extent of aeration on growth of Escherichia coli DH5α (ATCC 53868) in a 1L bioreactor at 37 oC with LB Lennox growth medium was investigated. Experiment results revealed that E. coli DH5α growth in bioreactor at 1 VVM aeration with 1 g/L PEG was faster than that in a 250 mL glass shake flask, and with greater secretion of alkaline metabolites. Similar optical density obtained between bioreactor and shake flask cultivation pointed to the maximized utilization of growth medium nutrients for biomass formation. Increase in bioreactor aeration to 3 VVM at 1 g/L PEG, however, resulted in increased secretion of acidic metabolites into the culture broth while allowing similar maximal optical density to be obtained compared to aeration of 1 VVM at 1 g/L PEG. This indicated that E. coli DH5α was able to adapt to physiological impacts from increased aeration and highlighted that no significant metabolic energy was diverted from biomass formation. Finally, increase in PEG concentration to 10 g/L from 1 g/L did not introduce additional toxicity effect given that growth profile of E. coli DH5α under the two PEG concentrations overlapped each other. However, observations of reduced secretion of acidic metabolites at the outset of growth in 10 g/L PEG pointed to physiological impacts that did not affect growth rates and biomass formation. Collectively, E. coli DH5α was able to tolerate enhanced aeration of 3 VVM and 10 g/L PEG anti-foam without significant detrimental impacts on growth rates and biomass formation.

scholarly journals Effect of different aeration and polyethylene glycol concentration on growth of Escherichia coli DH5α in a 1L bioreactor

2018 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Polyethylene Glycol ◽  
Cell Growth ◽  
Growth Medium ◽  
Growth Rates ◽  
Shake Flask ◽  
Foam Formation ◽  
Biomass Formation ◽  
E Coli ◽  
Toxicity Effect

Large culture volume in bioreactor necessitates aeration for providing sufficient oxygen for cell growth. Thus, extend of aeration and amount of anti-foam needed for suppressing foam formation are key parameters determining the success of bioreactor fermentation. Specifically, while aeration provides more oxygen for powering cellular metabolism and could lead to faster growth rate and more efficient metabolism, it also introduces greater shear stress, mixing and foam formation. On the other hand, anti-foaming agents such as polyethylene glycol (PEG) could exert a toxicity effect on cells as well as introducing increased osmolarity and viscosity that could hamper cell growth. In this preliminary study, effect of different PEG concentrations and extent of aeration on growth of Escherichia coli DH5α (ATCC 53868) in a 1L bioreactor at 37 oC with LB Lennox growth medium was investigated. Experiment results revealed that E. coli DH5α growth in bioreactor at 1 VVM aeration with 1 g/L PEG was faster than that in a 250 mL glass shake flask, and with greater secretion of alkaline metabolites. Similar optical density obtained between bioreactor and shake flask cultivation pointed to the maximized utilization of growth medium nutrients for biomass formation. Increase in bioreactor aeration to 3 VVM at 1 g/L PEG, however, resulted in increased secretion of acidic metabolites into the culture broth while allowing similar maximal optical density to be obtained compared to aeration of 1 VVM at 1 g/L PEG. This indicated that E. coli DH5α was able to adapt to physiological impacts from increased aeration and highlighted that no significant metabolic energy was diverted from biomass formation. Finally, increase in PEG concentration to 10 g/L from 1 g/L did not introduce additional toxicity effect given that growth profile of E. coli DH5α under the two PEG concentrations overlapped each other. However, observations of reduced secretion of acidic metabolites at the outset of growth in 10 g/L PEG pointed to physiological impacts that did not affect growth rates and biomass formation. Collectively, E. coli DH5α was able to tolerate enhanced aeration of 3 VVM and 10 g/L PEG anti-foam without significant detrimental impacts on growth rates and biomass formation.

scholarly journals Effect of polyethylene glycol on growth of Escherichia coli DH5α and Bacillus subtilis NRS-762

2020 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Polyethylene Glycol ◽  
Optical Density ◽  
Culture Broth ◽  
Dependent Manner ◽  
Biomass Formation ◽  
E Coli ◽  
Toxicity Effect ◽  
Dose Dependent

AbstractPolyethylene glycol is commonly used in fermentation as an anti-foam for preventing the rise of foam to the top plate of the bioreactor, which increases contamination risk. However, its potential toxicity to growth of various microorganisms is not well understood at the species and strain level. Hence, the objective of this study was to understand the impact of different concentrations of polyethylene glycol at the 1, 5 and 10 g/L level on the aerobic growth of Escherichia coli DH5α and Bacillus subtilis NRS-762 in LB Lennox medium in shake flasks. Experiment results revealed that polyethylene glycol (PEG) (molecular weight ∼8000 Da), at all concentrations tested, did not affect biomass formation and metabolism in E. coli DH5α at 37 °C. This came about through the observation of similar maximal optical density obtained during growth of E. coli DH5α under differing concentrations of PEG. Furthermore, the anti-foam did not affect the pH profile. On the other hand, PEG did exhibit some toxicity towards the growth of B. subtilis NRS-762 in LB Lennox medium. Specifically, maximal optical density obtained decline with higher exposure to PEG in a concentration dependent manner, up to a threshold concentration of 5 g/L. For example, maximal optical density obtained in B. subtilis NRS-762 without addition of PEG was 4.4, but the value obtained with 1 g/L of the anti-foam decreased to 4.1 and a further 3.8 on exposure to 5 g/L and 10 g/L PEG. pH variation in culture broth, however, told a different story, where the profiles for exposure to PEG at all concentrations coincide with each other and was similar to the one without exposure to the anti-foam; thereby, suggesting that metabolic processes in B. subtilis NRS-762 were not significantly affected by exposure to PEG. Collectively, PEG anti-foam exerted species-specific toxicity effect on biomass formation, and possibly metabolism. The latter may not be sufficiently significant to affect the types of metabolites secreted by the bacterium, and thus, be detected by measurement of pH of culture broth. E. coli DH5α was better able to cope with PEG at all concentrations compared to B. subtilis NRS-762, which showed dose-dependent toxicity effect on biomass formation.HighlightsPolyethylene glycol (molecular weight ∼ 8000 Da) did not affect aerobic growth of Escherichia coli DH5α at 37 °C in LB medium at all concentrations tested: 0, 1, 5, 10 g/L.Growth curves of the bacterium at different concentrations of polyethylene glycol (PEG) coincided with each other.Similar pH profiles were also obtained for E. coli DH5α growth in LB medium with different PEG concentrations.However, PEG exerted toxicity effect on Bacillus subtilis NRS-762 during growth in LB medium at 30 °C, with reduction of biomass formation in a dose dependent manner.Similar to the case for E. coli DH5α. pH profiles of B. subtilis NRS-762 coincided with each other irrespective of the concentrations of PEG used.

scholarly journals Staphylococcus aureus MazF Specifically Cleaves a Pentad Sequence, UACAU, Which Is Unusually Abundant in the mRNA for Pathogenic Adhesive Factor SraP

2009 ◽  
Vol 191 (10) ◽  
pp. 3248-3255 ◽  
Author(s):  
Ling Zhu ◽  
Koichi Inoue ◽  
Satoshi Yoshizumi ◽  
Hiroshi Kobayashi ◽  
Yonglong Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Cell Growth ◽  
Bioinformatics Analysis ◽  
Human Tissues ◽  
Regulatory Relationship ◽  
E Coli ◽  
Pathogenic Factors ◽  
Inhibit Cell Growth ◽  
Adhesive Factor

ABSTRACT Escherichia coli mRNA interferases, such as MazF and ChpBK, are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems present in its genome. A MazF homologue in Staphylococcus aureus (MazFSa) has been shown to inhibit cell growth when induced in E. coli. Here, we determined the cleavage site for MazFSa with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. MazFSa specifically cleaves the RNA at a pentad sequence, U↓ACAU. Bioinformatics analysis revealed that this pentad sequence is significantly abundant in several genes, including the sraP gene in the S. aureus N315 strain. This gene encodes a serine-rich protein, which is known to play an important role in adhesion of the pathogen to human tissues and thus in endovascular infection. We demonstrated that the sraP mRNA became extremely unstable in comparison with the ompA mRNA only when MazFSa was induced in E. coli. Further bioinformatics analysis indicated that the pentad sequence is also significantly abundant in the mRNAs for all the pathogenic factors in S. aureus. This observation suggests a possible regulatory relationship between the MazEFSa TA module and the pathogenicity in S. aureus.

scholarly journals In Vivo Modulation of a DnaJ Homolog, CbpA, by CbpM

2007 ◽  
Vol 189 (9) ◽  
pp. 3635-3638 ◽  
Author(s):  
Matthew R. Chenoweth ◽  
Nancy Trun ◽  
Sue Wickner
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Stationary Phase ◽  
Specific Inhibitor ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
E Coli ◽  
Hsp70 Chaperone

ABSTRACT CbpA, an Escherichia coli DnaJ homolog, can function as a cochaperone for the DnaK/Hsp70 chaperone system, and its in vitro activity can be modulated by CbpM. We discovered that CbpM specifically inhibits the in vivo activity of CbpA, preventing it from functioning in cell growth and division. Furthermore, we have shown that CbpM interacts with CbpA in vivo during stationary phase, suggesting that the inhibition of activity is a result of the interaction. These results reveal that the activity of the E. coli DnaK system can be regulated in vivo by a specific inhibitor.

scholarly journals Rapid Growth of UropathogenicEscherichia coliduring Human Urinary Tract Infection

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Valerie S. Forsyth ◽  
Chelsie E. Armbruster ◽  
Sara N. Smith ◽  
Ali Pirani ◽  
A. Cody Springman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Urinary Tract ◽  
Growth Rates ◽  
High Growth ◽  
Replication Forks ◽  
Chromosomal Replication ◽  
E Coli ◽  
Tract Infections

ABSTRACTUropathogenicEscherichia coli(UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenicE. coli(ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than otherE. coliisolates and survive in that niche. To date, there has not been a reliable method available to measure their growth ratein vivo. Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robustin vivo, matching or exceedingin vitrogrowth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth ratesin vivoat 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR,E. coliin urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth ratesin vivoand resistance to the innate immune response appear to be critical phenotypes of UPEC strains.IMPORTANCEUropathogenicEscherichia coli(UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized byE. colito colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measurein vivogrowth rates of other bacterial pathogens during host colonization.

scholarly journals Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

2010 ◽  
Vol 76 (14) ◽  
pp. 4655-4663 ◽  
Author(s):  
Sean M. Lee ◽  
Aaron Wyse ◽  
Aaron Lesher ◽  
Mary Lou Everett ◽  
Linda Lou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Bacterial Species ◽  
Intestinal Flora ◽  
Average Density ◽  
Host Bacterium ◽  
E Coli ◽  
K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

scholarly journals Laboratory-Dependent Bacterial Ecology: a Cautionary Tale

2006 ◽  
Vol 72 (4) ◽  
pp. 3032-3035 ◽  
Author(s):  
K. J. O'Keefe ◽  
N. M. Morales ◽  
H. Ernstberger ◽  
G. Benoit ◽  
P. E. Turner
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Equilibrium Composition ◽  
Cautionary Tale ◽  
Bacterial Strains ◽  
E Coli ◽  
Bacterial Ecology ◽  
Rule Out

ABSTRACT Although laboratory dependence is an acknowledged problem in microbiology, it is seldom intensively studied or discussed. We demonstrate that laboratory dependence is real and quantifiable even in the popular model Escherichia coli. Here laboratory effects alter the equilibrium composition of a simple community composed of two strains of E. coli. Our data rule out changes in the bacterial strains, chemical batches, and human handling but implicate differences in growth medium, especially the water component.

scholarly journals Different Cellular Origins and Functions of Extracellular Proteins from Escherichia coli O157:H7 and O104:H4 as Determined by Comparative Proteomic Analysis

2016 ◽  
Vol 82 (14) ◽  
pp. 4371-4378 ◽  
Author(s):  
Nazrul Islam ◽  
Attila Nagy ◽  
Wesley M. Garrett ◽  
Dan Shelton ◽  
Bret Cooper ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Medium ◽  
Foodborne Pathogen ◽  
The United States ◽  
Extracellular Proteins ◽  
Growth Media ◽  
Environmental Matrices ◽  
Content Type ◽  
E Coli ◽  
Relative Abundances

ABSTRACTExtracellular proteins play important roles in bacterial interactions with the environmental matrices. In this study, we examined the extracellular proteins fromEscherichia coliO157:H7 and O104:H4 by tandem mass spectrometry. We identified 500 and 859 proteins from the growth media ofE. coliO157:H7 and O104:H4, respectively, including 371 proteins common to both strains. Among proteins that were considered specific toE. coliO157:H7 or present at higher relative abundances in O157:H7 medium, most (57 of 65) had secretion signal sequences in their encoding genes. Noticeably, the proteins included locus of enterocyte effacement (LEE) virulence factors, proteins required for peptidyl-lipoprotein accumulation, and proteins involved in iron scavenging. In contrast, a much smaller proportion of proteins (37 of 150) that were considered specific to O104:H4 or presented at higher relative abundances in O104:H4 medium had signals targeting them for secretion. These proteins included Shiga toxin 2 subunit B and O104:H4 signature proteins, including AAF/1 major fimbrial subunit and serine protease autotransporters. Most of the abundant proteins from the growth medium ofE. coliO104:H4 were annotated as having functions in the cytoplasm. We provide evidence that the extensive presence of cytoplasmic proteins inE. coliO104:H4 growth medium was due to biological processes independent of cell lysis, indicating alternative mechanisms for this potent pathogen releasing cytoplasmic contents into the growth milieu, which could play a role in interaction with the environmental matrices, such as pathogenesis and biofilm formation.IMPORTANCEIn this study, we compared the extracellular proteins from two of the most prominent foodborne pathogenicE. coliorganisms that have caused severe outbreaks in the United States and in Europe.E. coliO157:H7 is a well-studied Shiga toxigenic foodborne pathogen of the enterohemorrhagic pathotype that has caused numerous outbreaks associated with various contaminated foods worldwide.E. coliO104:H4 is a newly emerged Shiga toxigenic foodborne pathogen of the enteroaggregative pathotype that gained notoriety for causing one of the most deadly foodborne outbreaks in Europe in 2011. Comparison of proteins in the growth medium revealed significant differences in the compositions of the extracellular proteins for these two pathogens. These differences may provide valuable information regarding the cellular responses of these pathogens to their environment, including cell survival and pathogenesis.

Kinetics and modelling of batch fermentation for the production of organic solvent tolerant and thermostable lipase by recombinant E. coli / Organik çözücü toleranslı ve ısıya dayanıklı rekombinan E. coli lipaz üretiminin kinetiği ve grup fermentasyonu modellemesi

2015 ◽  
Vol 40 (4) ◽  
Author(s):  
Arbakariya Bin Ariff ◽  
Rubina Nelofer ◽  
Raja Nor Zaliha Raja Abdul Rahman ◽  
Mahiran Basri
Keyword(s):  
Cell Growth ◽  
Shake Flask ◽  
Lipase Production ◽  
Stirred Tank ◽  
Thermostable Lipase ◽  
Stirred Tank Bioreactor ◽  
Monod Equation ◽  
E Coli ◽  
Organic Solvent Tolerant ◽  
Solvent Tolerant

AbstractObjective: Kinetics of organic solvent tolerant and thermostable lipase production by recombinant E. coli in shake flask level and 2 L stirred tank bioreactor level was studied to observe the variations of important kinetic parameters at two different levels of bioprocess.Methods: Unstructured models based on Monod equation for growth and Luedeking-Piret equation for lipase production and glucose consumption were used to predict cell growth, lipase production and glucose utilization. The shake flask fermentation experiments were carried out at different initial glucose and yeast extract concentrations using recombinant bacterial strain E. coli BL21. Lipase production was also carried out using 2L stirred tank bioreactor for comparison.Results: In all cases, the data fitted well to the proposed models. The highest growth and lipase activity were obtained at 25 g/L glucose and 25 g/L yeast extract. Cell growth (6.42 g/L) and lipase production (65.32 IU/mL) in 2 L stirred tank bioreactor was comparable to those obtained in shake flask fermentations. The calculated value of growth associated constant (9.874 IU/g/h) was much higher than that of non-growth associated constant (0.022 IU/g/h) in bioreactor as well as in shake flasks. The values of maximum specific growth rate (μm) and glucose saturation constant (KS) for shake flask fermentations, calculated from Monod equation, were 0.476 h-1 and 5.237 g/L respectively.Conclusion: From the modelling exercise, it was concluded that the lipase production is dominantly growth associated process. The kinetic parameter values for fermentations in shake flask and 2L stirred tank bioreactor were comparable, indicating that the bioprocess could be transferred into larger scale.

scholarly journals Possible epigenetic influence on cellular differentiation and lineage segregation in Escherichia coli

2019 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Cell Growth ◽  
Single Cell ◽  
Cell Fate ◽  
Cellular Differentiation ◽  
Environmental Stressors ◽  
Epigenetic Markers ◽  
E Coli ◽  
Growth Chambers

Epigenetics provides the critical connection between environmental influence and gene expression, where environmental stressors could modulate expression of specific genes in particular scenarios using molecular markers etched at the genome level. Hence, epigenetics likely play important roles in potentiating the development of specific lineages, cell fate or cellular differentiation. For example, when specific environmental stressor is present, epigenetic markers in the genome receive a signal for either activating or deactivating expression of particular sets of genes, which may be linked to the developmental trajectory of the organism. Using Escherichia coli as model organism, a possible study may investigate the role of epigenetics in influencing cellular differentiation of the bacterium. Specifically, a single E. coli cell would be propagated into a consortium of 12 or more bacterial cells in a microfluidics growth chamber. Genetic material extracted would be sent for single cell genomics, transcriptomics, and chromatin immunoprecipitation sequencing (ChIP-seq). After profiling, the residual population would be diverted by microchannels to 6 different cell growth chambers, where they would be cultivated under identical conditions for understanding possible triggers to cell differentiation. At suitable time points of 2, 4, 6, 8, 10, 12 hours, single cell would be extracted from each growth chamber for profiling single cell genomics, transcriptomics, and epigenetics markers. Optical and confocal laser scanning microscopy would provide readout of cell morphologies. Comparison of the readout between the original clonal population and those of the different growth chambers may provide important points for correlating epigenetic markers with gene expression and phenotypic readout in cell lineage, fate and differentiation. In subsequent experiments, different environmental stressors such as pH, imbalance nutrient composition between carbon and nitrogen, nanoparticles or heavy metals, could be used as triggers for specific cell growth response guided by epigenetic programmes embedded within the epigenome of the bacterium. Collectively, epigenetics hold influence for cellular differentiation in view of specific environmental stressors, where epigenetic markers on the genome communicate specific environmental factor's effect on the organism through altering expression of particular sets of genes, that result in different cell fate, lineage and differentiation. Using modern single cell techniques at the genomics, transcriptomics and epigenomics level, the study hopes to elucidate epigenetic potentiators of cellular differentiation in E. coli with and without environmental stressors such as nutrient deprivation, pH and toxic metals.

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