Abstract
BackgroundSmall-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.ResultsIn 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.ConclusionThus, 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.
Highlights
Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem
It is well known that oxygen limitations during bacterial fermentation can be deleterious to cell growth and productivity due to either diminished respiratory activity or the production of inhibitory byproducts [1]
Lee and coworkers demonstrated the E. coli nar promoter is maximally induced at microaerobic oxygen levels (1–2% of air saturation, left panel)
Summary
Small-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. While it's clear that low bulk oxygen concentration (either in the entire tank, or in particular regions of the tank) can lead to oxygen limitations, what is often not well understood is that significant oxygen limitations can occur even when the bulk dissolved oxygen concentration is well above the critical level This phenomenon occurs as a result of fluid segregation, or incomplete micromixing. The concept of fluid segregation, or micromixing, was first described nearly 50 years ago [2,3], has become a part of classical chemical-reaction engineering [4], and appears in a number of textbooks [5,6,7] Those conducting fermentations often think primarily in terms of macromixing, or movement of bulk fluid throughout the bioreactor. While extensive studies have shown that the degree of micromixing can impact chemical reactors [11], a limited number of studies have shown that it can significantly impact bioreactors growing bacteria [12], yeast[13,14] and filamentous fungi [15,16]
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