MICRO-ORGANISMS BASED BIOSENSORS 4 ENVIRONMENTAL MONITORING

Microorganism-based biosensors. Microorganism-based biosensors tend to use one of three primary mechanisms. For the first mechanism, the pollutant is a respiratory substrate. Biosensors that detect biodegradable organic compounds measured as biological oxygen demand (BOD) are the most widely reported of the microorganism-based biosensors using this mechanism. Several of these devices are commercially available from vendors including: Nisshin Electric Co. Ltd., Tokyo; Autoteam, GmbH, Berlin; Prufgeratewrk, Medingen GmbH, Dresden; and Dr. Lange, GmbH, Berlin. The use of these devices has been incorporated into industrial standard methods in Japan.^24-25

Biological oxygen demand is widely used as an indicator of the amount of biodegradable organic compounds found in industrial waste water. The standard procedure (termed BOD₅) involves a 5 day incubation of the environmental or industrial water sample with an inoculum of microorganisms typically present in the waste treatment system to yield an endpoint measurement for oxygen consumption.²⁵ The use of indicator microorganisms interfaced to signal transducers allows the measurement of the rate of organic compound metabolism rather than an endpoint; thus, data can be acquired in a significantly shorter time frame (e.g., 15 min to 1 hr), rendering this technology highly advantageous for process control applications. Although these biosensors appear to work well for in situ monitoring of industrial waste waters that result in high BOD values, they currently require improvements in several areas. The primary limitations for these methods involve the variability encountered in calibration of the biosensor response to BOD₅ values. This arises from the fact that specific microbial species (used in biosensors) have characteristic substrate spectra which may or may not correspond well with the spectrum of compounds present in the sample. Additional variability results from the presence of polymers (such as protein, starch, and lipid) which must be broken down to monomers before they can be metabolized; this changes the linearity of the response over time and can make interpretation of the result problematic.

Current progress on these technologies involves several areas. These areas include: the acid-induced breakdown of biological polymers prior to biosensor analysis, the selection of microorganisms with broad substrate spectra, and the introduction of novel transduction techniques. In addition, a recent report exploring the feasibility of disposable BOD sensors suggests considerable promise for advancement in this area.²⁶

Another mechanism used for microorganism-based biosensors involves the inhibition of respiration by the analyte of interest. Microbial respiration and its inhibition by various environmental pollutants have been measured both optically^27-28 and electrochemically.²⁹ Inherent advantages of these techniques apply primarily to the use of microorganisms as compared to isolated enzymes.²⁴ Microorganism-based biosensors are relatively inexpensive to construct and can operate over a wide range of pH and temperature. General limitations involve the long assay times including the initial response and return to baseline. These characteristics are primarily determined by the cellular diffusion characteristics that can be modified by using genetically engineered microorganisms. The broad specificity of these biosensors to environmental toxins may be an advantage or disadvantage depending on the intended application. In this respect, these devices might be most applicable for general toxicity screening or in situations where the toxic compounds are well defined, or where there is a desire to measure total toxicity through a common mode of action.

Biosensors have also been developed using genetically engineered microorganisms (GEMs) that recognize and report the presence of specific environmental pollutants. The microorganisms used in these biosensors are typically produced with a constructed plasmid in which genes that code for luciferase or -galactosidase are placed under the control of a promoter that recognizes the analyte of interest. Because the organism's biological recognition system is linked to the reporting system, the presence of the analyte results in the synthesis of inducible enzymes which then catalyze reactions resulting in the production of detectable products. With respect to environmental applications, the primary disadvantage for this type of biosensor is the limited number of GEMs which have been constructed to respond to specific environmental pollutants. Nevertheless, reported advances include the development of GEMs involving a variety of bioremediation pathways and mechanisms. GEMs that could report both the metabolic condition of the relevant microorganisms as well as the rates of pollutant breakdown could be very useful.