Masters Theses

Bo Huang

Date of Award

8-1992

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

T. W. Wang, G.Sayler

Committee Members

Robert Burlage, Bert Lampson

Abstract

During the operation of any bioreactor process, the most crucial elements are the microbial biomass and their metabolic state of growth. One of the major inhibiting factors in the development and implementation of sophisticated bioreactor control regimes has been the lack of suitable on-line biosensors for measuring such parameters. The objective of the current work is to develop and evaluate a novel on-line biosensor based on the light emission capabilities of a recombinant host cell used in the bioreactor process. The cells are genetically engineered to include the lux gene operon, originally isolated from bioluminescent marine bacteria, such as Vibrio fischeri. The luxAB gene product is the enzyme luciferase, which catalyzes intracellular reactions leading to the emission of visible light at 490 nm wavelength. The focus of this work was to develop a correlation between the intensity of emitted light from a prototype E. coli culture and the corresponding cell biomass concentration. The bioluminescence was to be monitored to give inferred measurement of the cell biomass concentration in a bioreactor process. A qualitative correlation between the intensity of emitted light and the cell biomass shows that bioluminescence appears to be a reflection of the relative rate of growth rather than the absolute amount of biomass, in that the maximum light emission always coincides with the point of maximum growth rate. Due to the inconsistency in the emitted light intensity from batch to batch, a consistent quantitative correlation was not successful. However, with the inconsistency removed by normalization, correlation results suggest that the light intensity variation from batch to batch may be an artifact rather than inherent. We predict that a more consistent correlation in continuous culture runs may be expected in the future.

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