Doctoral Dissertations

Date of Award

5-1997

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemical Engineering

Major Professor

Paul Bienkowski, Gary Sayler

Committee Members

Prados, White, Phelps

Abstract

Bioreporters are bacteria that have been genetically engineered to emit light (bioluminescence). Bioluminescence is known to be biochemically related to induction of the fher operon and corresponding production of the lax system enzymes that catalyze the light producing reactions. Substrates for these reactions include oxygen, aldehyde and the electron transport system components FMNH, NADH, and ATP. The traditional use of bioluminescence has been as a signal of transcription under highly controlled experimental conditions. Bioluminescent reporters offer an opportunity to extend the traditional uses of bioluminescence to engineering scale process design, monitoring and control. However, to extend the application of bioreporters, a quantitative relationship between bioluminescence and relevant environmental conditions needs to be established

Inducible and constitutive type bioluminescent reporters were examined in this research. The inducible reporters, Pseudomonas putida B2 and TVAR, contain the lux genes under toluene dioxygenase (rod) promoter control. Toluene induces both the fod and her operon. The toluene dioxygenase enzymes catalyze the degradation of toluene as well as the co-metabolism of trichloroethylene (TCE). The constitutive reporter, Shk 1, is an activated sludge microorganism containing a plasmid with the lux genes under the control of constitutive promoter.

Experiments were conducted under batch, semi-continuous and continuous cultivation conditions, and utilized batch, chemostar and turbidostat reactors. The primary conditions varied in these experiments were inducer, growth substrate and toxicant concentration. In limited experiments, pre-exposure to toluene, oxygen concentration, and growth source type were found to affect the bioluminescent response. Light was measured under steady-state and transient conditions with photonsultiplier technology.

A simple mechanistic mathematical model was developed that described hioduminescence and the degradation of toluene and TCE by the rod har reporters under batch and continuous cultivation conditions. The model incorporated (1) degradation dependence on inducer and growth stubstrate, co-metabolic substrate, and biomass concentrations; and (2) bioluminescence dependence on inducer and growth substrate, luciferase and biomass concentrations.

In both batch and continuous cultivation, bioluminescence increased at a rate greater than cell growth upon exposure to increased toluene. An accumulation of active luciferase molecules as well as in vivo enzyme decay mechanisms were proposed to mechanistically explain this behavior.

In tod-lux hioreporter chemostat experiments, bioluminescence transiently increased in response to increasing feed concentrations, then declined as steady-state was approached. The transient maximum hioluminescence corresponded with the soluene feed concentration due to increased steady-state cell densities. The transient increase in bioluminescence corresponded to increased toluene (growth substrate and inducer) availability in the reactor. Limiting growth substrate and possibly lack of induction of the tod operon was proposed to explain the decrease in light as the chemostat reached steady-state toluene concentration and cell density.

Bioluminescence from the Shk 1 reporter in a chemostat and a semi-continuous turbidostat reactor was compared. Steady-state bioluminescence for five different chemostat dilution rates was approximately the same (20 namp) and much lower than the steady-state bioluminescence from the turbidostat (160 namp). As the Shk 1 bioreporter does not require an inducer for light production, the difference in light was attributed to growth substrate availability

The bioluminescent response of the Shk 1 reporter to pH and the toxicants cadmium, 2,4-dinitrophenol, bydroquinone and ethanol was examined (0 to 10,000 mg/L). In batch and continuous sampling systems with the Shk 1 bioreporter bioluminescence declined more severely with increasing cadmium, 2,4-dinitrophenol, and lydroquinone concentration, while biolaminescence remained constant upon exposure to 10,000 mg/l. ethanol. The decrease in bioluminescence corresponded to the decrease in oxygen consumption rates of industrial activated sludge in response to hydroquinone exposure. Biosensors incorporating the Shk1 reporter were designed to contact the reporter with the process fluid to be sampled and were tested in bench scale activated sludge reactors. Additional growth substrate was required for the reporter to emit light. None of the designs tested provided a constant and significant level of bioluminescence

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