Non-destructive monitoring of microbial biofilms using on-line devices

Author

David E. Nivens

Date of Award

5-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

James Q. Chambers

Committee Members

Schell, Cook, David C. White

Abstract

Case studies establish that microbial biofilms affect the quality of water, facilitate the biodeterioration of surfaces, can cause infectious diseases, and can result in the malfunction of implanted medical devices. In response to these data, numerous studies that employ classical microbiological methods strive to gain insight into the formation and function of biofilms. However, sample removal as well as microbial growth on liquid or semi-solid growth media severely limit the ability of these procedures to detect and, to a greater extent, monitor bacteria on solid surfaces. To circumvent the problems inherent to these microbiological procedures, analytical methods are sought which provide on-line and non-destructive analyses of biofilms. This dissertation describes the evaluation, development, and application of online analytical methodologies using the quartz crystal microbalance (QCM) and Fourier transform infrared spectroscopy (ATR/FT-IR) to detect and monitor microbial biofilms in real-time. The QCM, which used 5 MHz AT-cut quartz crystals, measured frequency shifts caused by the attachment and growth of bacteria on the surface of the resonator. These QCM studies involved long-term monitoring of aqueous environments and encountered difficulties resulting from hydraulic pressure and temperature fluctuations. Frequency data showed that the temperature sensitivity for crystals mounted in flow cells and exposed to constant hydraulic pressure was greater than unstressed crystals exposed to air. Pressure changes at constant temperature linearly correlated with frequency shifts producing a slope of -2970 Hz•cm²kg^-1 Under sterile conditions at a constant pressure and temperature, the long-term drift noise of the QCM was determined to be ±. 4 Hz. When inoculated with Pseudomonas cepacia cells, the QCM monitored a frequency shift resulting from the attachment and growth of the bacteria on the surface of the resonator. At different frequency shift, the attached bacteria were enumerated by acridine orange staining and subsequent microscopic examination. From an empirically derived second order equation, the dose response curve plotting the frequency shift data versus the number of attached cells estimated the limits of detection to be 3 x 10⁵ P. cepacia cells/cm². Early experiments estimated the limits of detection for Caulobacter crescentus to be 3 X 10⁴ cells/ cm². Similarly, the ATR/FT-IR technique was used to monitor the initial formation, development, and physiology of P. cepacia and C. crescentus biofilms on Ge internal reflection elements (IRE) by detection of infrared adsorption bands. Plotting amide II absorbance versus the number of attached C. crescentus cells, the limit of detection was estimated to be 5 x 10⁵ cells/cm². This technique followed the initial formation of a biofilm and monitors the chemistry within approximately one μm of the solution/IRE interface. The technique was used to monitor the 1738 cm^-1 band which was established to be a biomarker for the synthesis of poly-β-hydroxyalkanoates, a carbon storage product. In addition, the depth of chemical sensitivity was increased by using ZnSe IREs. Spectra of C. crescentus biofilms on Ge IREs had lower carbonyl stretch to amide II band ratios than biofilms on ZnSe IREs, suggesting that the cell bodies may be oriented toward the flow stream.

Recommended Citation

Nivens, David E., "Non-destructive monitoring of microbial biofilms using on-line devices. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10741