Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors
Loading...
Permanent Link
Author/Creator
Author/Creator ORCID
Date
2009-01-15
Type of Work
Department
Program
Citation of Original Publication
Jose R Garcia, Hyung J Cha, Govind Rao, Mark R Marten and William E Bentley, Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors, Microbial Cell Factories 2009, 8:6 doi: 10.1186/1475-2859-8-6
Rights
This item is likely protected under Title 17 of the U.S. Copyright Law. Unless on a Creative Commons license, for uses protected by Copyright Law, contact the copyright holder or the author.
Attribution 2.0 Generic (CC BY 2.0)
Attribution 2.0 Generic (CC BY 2.0)
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 cellbased
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.