WERF Research Report Series Sensor Technology for Water Quality Monitoring

Two methods for the detection of important human pathogens, Cryptosporidium parvum and Helicobacter pylori, were investigated: a fiber optic biosensor, and real time PCR. The mechanism for specific detection in both methods is, recognition of specific DNA sequences in the target organisms. The Analyte 2000 was the biosensor that was used. Two methods for the detection of important human pathogens, Cryptosporidium parvum and Helicobacter pylori, were investigated: a fiber optic biosensor, and real time PCR. The mechanism for specific detection in both methods is recognition of specific DNA sequences in the target organisms. The biosensor that was used, the Analyte 2000, was originally developed for the detection of chemicals. It utilizes a fiber optic wave guide that propagates an evanescent light wave of very specific wavelength. The light excites fluorescent molecules bound to the waveguide, but not in the bulk solution, which theoretically enhances signal while reducing background interference. Attempts to develop this system for the detection of DNA were not successful due to poor detection of the target molecules. An assay analogous to a sandwich immunoassay was designed for use on the Analyte 2000. Specific oligonucleotide probes were designed to bind to the waveguides via biotin-streptavidin interaction, and were used to capture the target DNA. Pure target DNA representing unique genes in the organisms were synthesized by PCR. Detection of captured DNA was then attempted using an oligonucleotide detection probe designed to bind to the target. Two detection systems were employed: an indirect signal amplification system based on biotin-tyramide deposition, or direct detection of fluorescent signal from Cy-5 molecules. In all experiments performed there was very little difference between the signal generated with or without the target molecules. Many experiments were conducted to attempt to identify reasons for the poor signal. Signal was only of any significance when target amplicons were internally labeled with Cy-5 by PCR. Real time PCR as a method to detect the pathogens was also investigated. Though the PCR technique itself is very rapid, DNA extraction and purification requires preparation time. Filtration of up to one liter of well water, followed by concentration and "cleaning" Helicobacter pylori cells by immunomagnetic separation, was used to detect H. pylori seeded in a water source. Following cell lysis, the extracted DNA could be used directly in conventional PCR targeting the 16S rRNA gene to detect less than 265 cells per liter of water. DNA purification was not required for this level of detection. Initial studies to amplify lysed cells by real time PCR indicated that an incorrect product was made. When purified DNA was used for real time PCR, the correct product was produced from DNA representing as few as 100 cells. This publication can be purchased and downloaded via Pay Per View on Water Intelligence Online - click on the Pay Per View icon below

Biological wastewater treatment plants can be adversely affected by influent toxicity. The effects can range from poor clarifier biomass settling and elevated effluent BOD and ammonia levels to total plant kills. The goal of a research team was to create bioluminescent biosensors from the bacteria, found in biological wastewater treatment plants. Biological wastewater treatment plants can be adversely affected by influent toxicity. The effects can range from poor clarifier biomass settling and elevated effluent BOD and ammonia levels to total plant kills. These problems could be minimized or eliminated if an effective method existed for continuously monitoring biological wastewater treatment plant influent for toxicity to the treatment plant microorganisms. Current influent screening methods have not been proven to be adequate for adaptation to continuous screening in the field. The primary reasons include the batch-wise nature of the assays and an inadequate correlation between the assays and plant performance. The goal of the research team was to create new bioluminescent biosensors from different types of bacteria found in biological wastewater treatment plants for the development of a multi-channel continuous monitoring system. A system built from multiple biosensors would make it possible to differentiate between potential influent toxicity effects to different classes of bacteria (such as nitrifying and heterotrophic bacteria). The research team found it unexpectedly challenging to apply common microbiological transformation methods for laboratory strains to the wastewater treatment plant strains. The research team generated six new bioluminescent bioreporters from bacteria that are typical constituents of activated sludge. Of particular significance is a bioreporter developed using a Hyphomicrobium sp., which is a slow growing bacterium known to be present in significant numbers in some activated sludge plants. Of the six generated, initial bioluminescence and toxicity screening indicated that one strain (a Pseudomonad) was a particularly promising candidate due to its ease of cultivation and high light production. Further toxicity testing, however, determined that the response of the strain to 48 organic compounds and 8 metals commonly found in wastewater was similar to that of a previously created strain, Shk1 (also a Pseudomonad). Further work is therefore needed in the generation of appropriate biosensors and test conditions for populations not represented by the new heterotrophic biosensor.