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INVESTIGATING DIRECTED EVOLUTION AND GENETIC ENGINEERING WITH VITREOSCILLA HEMOGLOBIN TO PRODUCE CULTURES FOR LOW AERATION BIOLOGICAL WASTEWATER TREATMENT
Title
INVESTIGATING DIRECTED EVOLUTION AND GENETIC ENGINEERING WITH VITREOSCILLA HEMOGLOBIN TO PRODUCE CULTURES FOR LOW AERATION BIOLOGICAL WASTEWATER TREATMENT
Creator
Kunkel, Stephanie
Date
2014, 2014-07
Description
The dominance of hemoglobin (Hb)-expressing bacteria in biological wastewater treatment systems could improve oxygen utilization under low...
Show moreThe dominance of hemoglobin (Hb)-expressing bacteria in biological wastewater treatment systems could improve oxygen utilization under low dissolved oxygen (DO) conditions. Hb-proteins are versatile molecules that have several biological functions. Here, Nitrosomonas europaea has been transformed with various plasmids; of particular interest is a recombinant plasmid bearing the constitutive Amo1 promoter and the gene (vgb) encoding the hemoglobin from the bacterium Vitreoscilla. Expression of VHb was assayed using various visible spectral methods, and VHb production seen in this recombinant strain. There were several positive effects on N. europaea metabolism related to VHb expression that were seen, specifically the ability of cultures to convert ammonia to nitrite at a slightly higher rate as well as higher specific oxygen uptake rates (SOUR) at both high (near saturation, 7 mg O2/ L) and low (< 2 mg O2/L) dissolved oxygen (DO) conditions. In parallel to this, two activated sludge cultures from the same source were cultivated using synthetic wastewater seeded with activated sludge from the same source and were operated at high DO (near saturation) and low DO (0.25 mg O2/L) concentrations for 370 days. There were significant changes in the bacterial species and phyla present in each of the cultures at various time points during the 370 day operational period. In the low DO culture, over time, there was a much greater expression of single domain and truncated Hbs which may enhance utilization and delivery of oxygen to various enzymes as well as to the respiratory chain. A larger increase in heme b was also observed which coincides with this observation. By the end of the acclimation period, the SOUR values were about 30% greater in the low DO culture compared to the high DO culture. This indicated the successful adaption of the low DO culture to respire more efficiently and eventually outperform the high DO culture.
Ph.D. in Biology, July 2014
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EFFECTS OF ENGINEERING USING BACTERIAL HEMOGLOBIN ON THE ABILITY OF ETHANOLOGENIC E. COLI TO FERMENT LIGNOCELLULOSIC HYRDOLYSATE TO ETHANOL
Title
EFFECTS OF ENGINEERING USING BACTERIAL HEMOGLOBIN ON THE ABILITY OF ETHANOLOGENIC E. COLI TO FERMENT LIGNOCELLULOSIC HYRDOLYSATE TO ETHANOL
Creator
Kunkel, Stephanie
Date
2011-05-03, 2011-05
Description
E. coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol has been further engineered in our laboratory using...
Show moreE. coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol has been further engineered in our laboratory using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The resulting strain (TS3) expresses VHb at a moderate level under microaerobic conditions in shake flasks and produces more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the carbon source. In the work reported here, this research was extended, focusing particularly on fermentation of lignocellulosic hydrolysate. The increased ability of the VHb-bearing strain to produce ethanol from pure glucose or xylose was confirmed. A VHb-correlated increase in ethanol production was also noted for lignocellulosic hydrolysate produced in our laboratory, but not for ligncellulosic hydrolysate prepared by the National Laboratory for Renewable Energy (NREL). Experiments were performed at several hydrolysate levels for both prepartories, in an attempt to determine the cause of the difference. One possibility is differences in levels of toxic hydrolysis byproducts in the two preparations as well as increased sensitivity to these toxins due to VHb expression.
M.S. in Biology, May 2011
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