Thesis submitted to the Faculty of the
Virginia Polytechnic Institute and State University
in partial fulfillment of the requirements for the degree of
Master of Science
Dr. Brian J. Love, Chairperson
Dr. Nancy G. Love
Dr. Kathleen Meehan
A biosensor is proposed that contains bacteria that naturally effluxes potassium ions when threatened by electrophilic species.Pseudomonas aeruginosa is an activated sludge isolate and possesses the characteristic potassium efflux response. It has been immobilized in calcium alginate beads, photopolymer disks, and a thermally reversible gel in order to ultimately incorporate the immobilized system into a functional biosensor. The potassium efflux and cell viability were measured in the immobilized matrices.
Wastewater treatment is of utmost importance; however, processes are easily upset. Upsets can be caused by various electrophiles found in the environment, and can cause serious health effects to people or the environment downstream from an upset. Electrophiles can cause the activated sludge in wastewater treatment facilities to deflocculate, and untreated water can be lost downstream. Devising a detection system for proactively sensing electrophiles prior to an upset is an important complementary goal.
Immobilization systems have been evaluated including photopolymer coated alginate beads and sol gel coated alginate beads. The thermally reversible gel, NIPA-co- AAc (N-isopropylacrylamide-co-acrylic acid), shows promise as an immobilization matrix for the bacteria; however its high lower critical solution temperature (LCST) of ~33oC is problematic for typical, ambient applications. Another thermally reversible copolymer, N-isopropylacrylamide-co-N-acryloyl-6-amino caproic acid (NIPA-co- AcACA) was synthesized; however, it did not form a continuous matrix; making it
useless as an immobilization scheme for biosensors. Alginate beads fall apart easily in bacteria media, but are structurally stable in potassium solutions. Cells immobilized in alginate beads seemed to efflux four times less potassium than did planktonic controls, while cells in thermally reversible gels effluxed a comparable amount of potassium as planktonic controls. This result may indicate a tighter matrix around the alginate immobilized cells, not allowing proper diffusion of potassium out of the matrix.
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