Limits...
Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors.

Garcia JR, Cha HJ, Rao G, Marten MR, Bentley WE - Microb. Cell Fact. (2009)

Bottom Line: The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation.However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation.As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, MD 21052, USA. marten@umbc.edu.

ABSTRACT

Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency - one verses four impellers.

Results: In both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen (DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space.

Conclusion: Thus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.

No MeSH data available.


Gene map of recombinant plasmid pNar-GFPuv. gfpuv gene is regulated by nar (Pnar) promoter.
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Figure 2: Gene map of recombinant plasmid pNar-GFPuv. gfpuv gene is regulated by nar (Pnar) promoter.

Mentions: We then used these two impeller configurations to carry out triplicate fed-batch fermentations with E. coli harboring our pNar-GFPuv plasmid (Figure 2). As noted above, all fermentations were run batchwise until the initial glucose concentration (20 g L-1) was consumed to less than 1 g L-1 (also marked by a rapid increase in %DO). Results are shown in Figure 3. Depicted data are the averages from three separate fermentations; the error bars denote standard deviation. We note that traditional measurements (glucose profile, OD600, and DO) all show nearly identical behavior. However the GFP trends (Figure 3B) show significantly higher expression when only one impeller is used versus four. This is represented even more dramatically in Figure 3C where specific GFP expression rose significantly higher with one versus four impellers.


Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors.

Garcia JR, Cha HJ, Rao G, Marten MR, Bentley WE - Microb. Cell Fact. (2009)

Gene map of recombinant plasmid pNar-GFPuv. gfpuv gene is regulated by nar (Pnar) promoter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2630296&req=5

Figure 2: Gene map of recombinant plasmid pNar-GFPuv. gfpuv gene is regulated by nar (Pnar) promoter.
Mentions: We then used these two impeller configurations to carry out triplicate fed-batch fermentations with E. coli harboring our pNar-GFPuv plasmid (Figure 2). As noted above, all fermentations were run batchwise until the initial glucose concentration (20 g L-1) was consumed to less than 1 g L-1 (also marked by a rapid increase in %DO). Results are shown in Figure 3. Depicted data are the averages from three separate fermentations; the error bars denote standard deviation. We note that traditional measurements (glucose profile, OD600, and DO) all show nearly identical behavior. However the GFP trends (Figure 3B) show significantly higher expression when only one impeller is used versus four. This is represented even more dramatically in Figure 3C where specific GFP expression rose significantly higher with one versus four impellers.

Bottom Line: The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation.However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation.As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, MD 21052, USA. marten@umbc.edu.

ABSTRACT

Background: Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency - one verses four impellers.

Results: In both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen (DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space.

Conclusion: Thus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.

No MeSH data available.