Introduction
In developing bioremediation technology or assessing the effectiveness of natural attenuation processes, it is ul¬ timately necessary to provide quantitative in situ measures of the active microbial biodegradative processes involved. This necessity is determined by several factors including: □ Proving that the bioremediation technology or microorganisms are effective in the contaminated environment □ Optimizing the bioremediation technology through process monitoring and control to maximize or sus- tain biodegradation processes under dynamic in situ conditions □ Evaluating the reduction in chemical contaminant levels for endpoint determination to evaluate ex- posure to bioavailable contaminants and metabolic transformation products The need to provide evidence documenting these factors can be met in some cases by conventional microbiological assessments or analytical chemical techniques, such as GC/MS, or with newer geohydrological methods, such as stable isotope ratios and push-pull well tests. However, often times these methods may lead to ambiguous results or are costly and time consuming in their environmental im¬ plementation. Recently new molecular biology based technology focusing on the level and activity of specific organisms and genes involved in the biodegradation process have been developed and applied for in situ analysis of bioremedia¬ tion technology and natural attenuation processes. Among these technologies is the analysis of messenger RNA (mRNA) levels indicative of expression of bio¬ degradative genes in situ, and the use of bioluminescent (lux) reporter gene technology for on-line real-time analysis of biodegradative expression and chemical sensing in con- taminated environments.  RNA analysis is a powerful tool for environmental assessment and, with development of microarray methods, can significantly enhance our under- standing of in situ microbial biodegradation processes. Gene expression monitoring using mRNA has been recently reviewed 1,2) and, while the technology continues to ad¬ vance, its complexity in application has somewhat limited its environmental utility. However, lux based bioreporter gene expression tech¬ nology has moved forward as a significant analytical tool in environmental chemical sensing and bioremediation process monitoring. It is the objective of this report to describe the recent developments and applications of this technology in bioremediation. These developments in¬ clude the real-time in situ analysis of chemical con¬ taminants, as well as biodegradation process analysis. Most recently living cell bioreporter technology has been incorporated into microelectronic integrated circuits sen¬ sor technology ultimately to provide wireless real-time monitoring technology for bioremediation.
Reporter Genes Systems
Bioreporters refer to intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in their environment (Fig. 1). Bioreporters contain two es¬ sential genetic elements, a promoter gene and a reporter gene.  The promoter gene is turned on (transcribed) when the target agent is present in the cell's environment. The promoter gene in a normal bacterial cell is linked to other genes that are then likewise transcribed and then translated into proteins that help the cell in either combating or adapting to the agent to which it has been exposed.  In the case of a bioreporter, these genes, or portions thereof, have been removed and replaced with a reporter gene. Conse- quently, turning on the promoter gene now causes the reporter gene to be turned on. Activation of the reporter gene leads to production of reporter proteins that ulti- mately generate some type of a detectable signal. There- fore, the presence of a signal indicates that the bioreporter has sensed a particular target agent in its environment. Originally developed for fundamental analysis of factors affecting gene expression, bioreporters were early on ap- plied for the detection of environmental contaminants 21 and have since evolved into fields as diverse as medical di- agnostics, precision agriculture, food-safety assurance, process monitoring and control, and bio-microelectronic computing.  Their versatility stems from the fact that there exist a large number of reporter gene systems that are capable of generating a variety of signals. Additionally, reporter genes can be genetically inserted into bacterial, yeast, plant, and mammalian cells, thereby providing considerable functionality over a wide range of host vec- tors. Several types of reporter genes are available for use in the construction of bioreporter organisms, and the signals they generate can usually be categorized as either colorimetric, fluorescent, luminescent, chemiluminescent or electro- chemical. Although each functions differently, their end product always remains the same – a measurable signal that is proportional to the concentration of the unique chemical or physical agent to which they have been exposed.   In some instances, the signal only occurs when a secondary sub- strate is added to the bioassay (luxAB, Luc, and aequorin). For other bioreporters, the signal must be activated by an external light source (GFP and UMT), and for a select few bioreporters, the signal is completely self-induced, with no exogenous substrate or external activation being required ( luxCDABE ).  The following sections outline in brief some of the reporter gene systems available and their existing applications. Bacterial luciferase (Lux):   Luciferase is a generic name for an enzyme that catalyzes a light-emitting reaction. Lu- ciferases can be found in bacteria, algae, fungi, jellyfish, insects, shrimp, and squid, and the resulting light that these organisms produce is termed bioluminescence. In bacter- ia, the genes responsible for the light-emitting reaction (the lux genes) have been isolated and used extensively in the construction of bioreporters that emit a blue-green light with a maximum intensity at 490 nm (Fig. 2)49. Three variants of lux are available, one that functions at < 30°C, another at <37°C, and a third at <45°C. The lux genetic system consists of five genes, luxA, luxB, luxC, luxD, and luxE. Depending on the combination of these genes used, several different types of bioluminescent bioreporters can be constructed. luxAB bioreporters:  luxAB bioreporters contain only the luxA and luxB genes, which together are responsible for generating the light signal.  However, to fully complete the light-emitting reaction, a substrate must be supplied to the cell. Typically, this occurs through the addition of the chemical decanal at some point during the bioassay procedure.  Numerous luxAB bioreporters have been con¬ structed within bacterial, yeast, insect, nematode, plant, and mammalian cell systems. luxCDABE bioreporters: Instead of containing only the luxA and luxB genes, bioreporters can contain all five genes of the lux cassette, thereby allowing for a completely in¬ dependent light generating system that requires no ex¬ traneous additions of substrate or any excitation by an external light source. So in this bioassay, the bioreporter is simply exposed to a target analyte and a quantitative in¬ crease in bioluminescence results, often within less than one hour.  Due to their rapidity and ease of use, along with the ability to perform the bioassay repetitively in real-time and on-line, makes luxCDABE bioreporters extremely attrac¬ tive. Consequently, they have been incorporated into a diverse array of detection methodologies ranging from the sensing of environmental contaminants to the real-time monitoring of pathogen infections in living mice.  Table 1 illustrates the widespread application of luxCDABE bioreporters.
Detecting the Optical Siganature
Using light as the terminal indicator is advantageous in that it is an easily measured signal. Optical transducers such as photo-multiplier tubes, photodiodes, microchan- nel plates, or charge-coupled devices are readily available and can be easily integrated for environmental research. For example, a "down-hole" photomultiplier system was built to insert into subsurface bore holes to directly measure light from soils originally inoculated with Pseudomonas fluorescens HK44 1.5 ). Figure 3 provides a diagram repre- sentation of the photomultiplier devise with a direct pho¬ tographic recreation of luminescing soil from HK 44 in situ biodegradation process activity. As these usually consist of large, table-top devices, demand for smaller, portable light readers for remote monitoring has resulted in the development of battery-operated, hand-held photomul¬ tiplier units. Recently, The Center for Environmental Biotechnology and Oak Ridge National Laboratory have taken steps towards genuine miniaturization of optical transducers and have successfully developed integrated circuits capable of detecting bioluminescence directly from bioreporter organisms (Fig. 4) 6). These bioluminescent bioreporter integrated circuits (BBICs) consist of two main components; photodetectors for capturing the on-chip bioluminescent bioreporter sig¬ nals and signal processors for managing and storing in- formation derived from bioluminescence (Fig. 5, 6). Re- mote frequency (RF) transmitters can also be incorporated into the overall integrated circuit design for wireless data Sayler et al.
relay. Since the bioreporter and biosensing elements are completely self-contained within the BBIC, operational capabilities are realized by simply exposing the BBIC to the desired test sample. In addition to incorporation in a BBIC format, the whole-cell bioreporter matrix can also be immobilized on something as simple as an indicator test strip. In this fashion, a home water quality indicator, for example, could be developed to operate in much the same way as a home pregnancy test kit. An example of lux-based bioreporters in use: Monitoring environmental contaminants in a groun- dwater plume A groundwater research facility at Columbus Air Force Base, Mississippi was contaminated with a simulated jet fuel mixture consisting of naphthalene, toluene, ethylben- zene, and p-xylene 7 ).  Numerous multilevel sampling wells installed upstream and downstream of the contaminant source allowed for monitoring of the contaminants. Typically, water would be pumped up from designated wells and sent to an off-site laboratory for contaminant analysis using gas chromatography/mass spectrometry (GC/MS) techniques. GC/MS analysis is extremely sensi¬ tive and accurate, and is by far the best method available for detecting chemical contaminants in environmental sources. However, it also requires expensive and bulky instrumen- tation, a trained technician, the use of hazardous chemi- cals, and a significant allotment of time.   As an alternative, we proposed using bioreporters as sensors for the groun- dwater contaminants. Two bioreporters were used, Pseu- domonas fluorescens 5RL, a bioreporter for naphthalene, and Pseudomonas putida TVA8, a bioreporter for tolu- ene 8-9). Analysis occurred on-site, where bioreporters were simply combined with groundwater samples and allowed to incubate for a set time. Resulting bioluminescence was measured using a field portable photomultiplier unit in- terfaced to a laptop computer. Duplicate samples were sent to an off-site laboratory for GC/MS determination of toluene and naphthalene concentrations.  Bioluminescent bioreporters consistently predicted contaminant concen- trations within 50% of the GC/MS analytic measurements (Fig. 7). Although in this case not highly quantitative, bioluminescent bioreporters did provide a rapid, general assessment of contaminant presence within the groun- dwater aquifer, and established an overall snapshot of plume dynamics within a few hours of initial sampling at a cost of approximately 1/10 of that of GC/MS analysis.
Conclusions
Bioreporter technology will provide a robust, cost- effective, quantitative method for rapid and selective de- tection and monitoring of chemical and biological agents in applications as far ranging as medical diagnostics, preci- sion agriculture, environmental monitoring, food safety, and process monitoring and control.  Their attractiveness lies in the fact that they can often be implemented in real- time, on-line bioassays within intact, living cell systems, thus providing a unique and revolutionarily new perspec- tive on bacterial, plant, and mammalian physiology and intracellular interactions. In conjunction with advanced photonic detection technologies such as the BBIC, bioreporters are increasingly becoming important tools for noninvasive monitoring regimes, especially in animal model systems.  The monitoring of light requires less time and fewer animals than conventional methods, thus reducing the cost of obtaining biologically relevant data. Consequently, the study of infectious disease, tumor progression and metastasis, gene therapy, mammalian de¬ velopment, and many other areas in which animal models are used as predictors for the human response to therapy can be greatly simplified and accelerated. The same ideals apply in cases of environmental monitoring and food safety, where rapid and remote monitoring using BBIC devices can strategically pinpoint areas of biological haz- ard, whether in the form of biological warfare agents or pathogenic E. coli presence. Further advances in bio- reporter genetics and miniaturized optics will clearly im¬ pact future monitoring and detection strategies in these fields as well as a host of others.