Biosensing environmental pollution
Introduction
Bioremediation (i.e. the use of plants or microorganisms to clean up environmental pollution) has seen a surge of interest in recent years. Traditionally, the efficacy of bioremediation has been determined chemically, by measuring changes in total pollutant concentrations, usually complemented by chromatographic results (gas chromatography or gas chromatography-mass spectrometry). Recently, however, attempts have been made to use biosensors, especially microbial whole-cell biosensors, to monitor the rate of pollutant elimination [1, 2, 3, 4, 5, 6, 7].
Biosensors are defined as monitoring systems based on the use of biological organisms or biologically derived reactions. For example, whole organisms, such as Daphnia or small fish, are being used to monitor water toxicity and enzymes can be used as biosensors of oxygen or glucose. The main drawback of biosensors is that, because they depend on biological systems, they are less reproducible than chemical methods and the values obtained are usually relative and not absolute. However, they can be considerably more time-effective and cost-effective and constitute a good tool for monitoring changes or on-line processes. Another important advantage of biosensors is that they detect only the biologically active pollutants, and the response is proportional to the level of toxicity.
This review focuses on whole-cell, ‘man-made’ biosensors based on recombinant DNA technology. These biosensors are constructed by fusing a pollutant-responsive gene promoter to a gene coding for a protein that can be easily quantified. In one such example, an Escherichia coli K-12 strain has been adapted for the detection of aromatic hydrocarbons. The E. coli cells carry the promoter of the xylS gene (induced by aromatic hydrocarbons) from Pseudomonas putida fused to a promoterless luciferase operon (from Vibrio harveyi or firefly). The expression of luciferase is induced by the presence of aromatic compounds and can be measured by light emission. Similar biosensors were constructed that can monitor the level of additional environmental pollutants, such as octane [3, 4].
Recently, new whole-cell biosensors have been reported in which gene expression is monitored by electrochemical means. These promoter-based, whole-cell biosensors are suitable for on-line and in situ monitoring of pollutants [8, 10, 11, 12]. An example is depicted in Figure 1, which shows a recombinant gene construct effective in biosensing heavy metals [8, 9]. In this construct, the promoter and N-terminal region of the zntA gene, involved in the efflux of heavy metals, is fused to a promoterless lacZ gene, which codes for the enzyme β-galactosidase. Bacteria carrying this gene fusion respond to the presence of heavy metals by inducing the production of β-galactosidase, an enzyme that can easily be monitored by colorimetric and electrochemical reactions.
Here, I review the major types of reporters and promoters used for the construction of biosensors and discuss the advantages and disadvantages of these different systems. I go on to look at possible future directions in biosensor research in relation to monitoring environmental pollution.
Section snippets
The construction of biosensors
As mentioned above, whole-cell, man-made biosensors typically consist of a promoter, responsive to one or more pollutants or toxicants, genetically fused to a promoterless reporter gene. The recombinant genes can be located on plasmids or on the chromosome. An effective biosensor depends on the correct choice of its two constituents: the promoter and the reporter gene.
In situ and on-line measurements
An important aspect of biosensing environmental pollutants is the ability to monitor in situ and preferably on-line. Having biosensors that can be used in the field dispenses with the need to bring samples to the laboratory, and enables real-time assessment of the level of pollution. On-line and in situ monitoring of gene expression can be performed by employing reporter enzymes whose activity can be monitored electrochemically [8]. The electrochemical measurements are highly sensitive,
Monitoring with biological systems – when and where
Biosensors are fast, cost-effective and indicate the level of ‘relevant’ toxic materials. Their main disadvantage is that they do not give absolute chemical values. Moreover, because they depend on biological systems, they can only indicate relative levels, never absolute levels. For authorities such as the Environmental Protection Agency, with the present set of regulations, the inability to determine absolute concentrations is a drawback. However, because there is no way to determine absolute
Future directions for biosensor development
There are several obvious directions for developing biosensors. One of the main problems with the use of biological material is the difficulty of maintaining constant activity for a long time. For environmental biosensors it is also important to enable long storage at room temperature. To meet these demands, various conservation techniques have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilization in biocompatible polymers of organic or inorganic
Conclusions
In conclusion, cell-based biosensors provide effective tools for detecting environmental pollution and toxicity. In the future, biosensors are likely to become smaller and more flexible and will enable the on-line and in situ monitoring of a large number of environmental parameters. Clearly, we are a long way from the times of the ‘classic’ biosensor — the canary down the mine shaft or the dog of the wine maker!
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported, in part, by the Manja and Morris Leigh Chair for Biophysics and Biotechnology.
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