The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:rhodes/vital:3944 |
| Date | January 2008 |
| Creators | Wutor, Victor Collins |
| Publisher | Rhodes University, Faculty of Science, Biochemistry, Microbiology and Biotechnology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | xxiii, 200 leaves, pdf |
| Rights | Wutor, Victor Collins |
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