Journal of Cleaner Production 11 (2003) 293–301 www.cleanerproduction.

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Biosensors as molecular tools for use in bioremediation
H.J. Purohit ∗
Environmental Modeling and Genomics Division, National Environmental Engineering Research Institute, Nehru Marg, Nagpur 440020, India Received 19 June 2001; accepted 20 May 2002

Abstract Biosensors are analytical tools, which use the biological specificity in sensing the target molecule. In this paper, the author reviews the design and application of molecular biosensors for use in bioremediation. The promoter selected from a genetic operon has been described as an interactive biological component for the target molecule to generate the signal. Different types of reporter systems have been described and as well as their application in tracking of levels of pollutants, nitrogen, phosphorus, dissolved oxygen in different habitats and toxic compounds. The paper emphasizes that in order to extend the applications of this scientific area, specialized research is needed in the aspects pertaining to bringing the biological recognition element into close proximity to the target molecules so that it can be integrated with the signal analysis system.  2002 Elsevier Science Ltd. All rights reserved.
Keywords: Biosensor; Bioremediation; Lux; GFP; Promoter

1. Introduction In the last decade, the biological sciences have been used in exciting new directions. As one result, the field of biotechnology has emerged as a discipline. Within that area, biosensors have become one of the newest dimensions of this revolution. Biosensor technology uses biological outputs to monitor processes. The signals from biosensors are picked up by microelectronic elements and processed through various types of processors. Bio-molecules, utilized as biosensors, are selective in their interaction with other molecules and the reactions always follow the same kinetics. This property of molecular specificity is used as the basis in designing biosensors. Therefore, a biosensor can be case-specific in its approach or with application of genetic engineering; more flexible biosensors can be also envisaged. The second component of this analytical device is a signalgenerating surface, which feeds the information flow to a signal-processing unit. There are different schematic diagrams available in the literature describing biosensors [1,2]. However, a biosensor is mostly case-specific

where the different components are interfaced; and are costume designed to accommodate the shelf life and stability of the biological component. Fig. 1 shows a more generalized scheme where the biological components, with the advent of genetic engineering, propose more flexible biosensors. This figure shows various options, through which the target molecule can be transformed, hydrolyzed or an enzymatic product. Similarly, light, temperature, and change in conductivity also could be one of the products of the biological reactions; and could be measured by the signals generated. 2. Biochemical potential and application to biosensor Biosensors are designed using a specific bio-active component for the desired conversion to yield a signal that can be monitored. Based on the component of the cells or the cell itself, there are various biological components that have been developed by different research groups that may be useful as biosensors. Whole cells may be used when a desired activity is targeted for expression of a recombinant protein and the reaction catalyzed through it. An example of this may be the potentiometric microbial electrode designed for detection of organophosphorous pesticides. This system uses the surface-expressed organophosphorous hydrolase [3].

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For this type of application. Therefore. Schematic depiction of target molecule is captured by ligand . In this article the author describes such biosensors. In such an environment. They are described as molecular biosensors since they use the promoters derived from specific genes to interact with target molecules. Another feature. Sometimes. hence they provide the required specificity in signal generation.294 H. Molecular biosensors The biological component in a molecular biosensor is a recombinant plasmid. . Bioremediation is an application of microbial capacity to transform complex organic molecules into simpler inorganic constituents. is separated. the additional requirement is a support system for the survival and multiplication of these designed sensing molecules. if the biological component through the rest of the environment. biosensors with the following basic features would be suitable. The promoters can be turned on or off with specific molecules. the enzymebased biosensors have been extensively explored. but still is kept sufficiently interactive. The case-specific recombinant plasmids can be genetically engineered so as to lead to a biosensor. Secondly. which to pass on the signal to processing unit to generate electronic output. Various enzymebased biosensors are reported for the detection of pesticides in different samples.J. There could be another option where the inherent property of a cell or tissue is used to generate the signal [4]. the specificity of monitoring towards the target molecule should be independent of changes in environmental conditions. It has a specific promoter. 1. whose expression is sensitive to a target molecule and uses the reporter system to generate the signal. it should not affect the microbial community structure if it is to be used as whole cells. The generation of signals is directly proportional to the expression of the promoter. Recent advances in clinical sample analysis show a trend where biosensors based on luminescence systems are used [6]. in such a . it is divided in a manner that the host chromosome carries a part of the supporting activity for expression of the promoter. interacting with the biological component to yield signal molecule. Of the different types of biosensors. or co-enzyme conversion. it represents an environment where the microbial community is thriving as they biodegrade various pollutants. The enzymes most often applied are cholinesterase and choline oxidase for the detection of organophosphorous pesticides [5]. Sometimes these reactions are superimposed with another biochemical event such as use of inhibition kinetics or coupling with other reaction(s). Biosensors using enzymes in their biological function generate the signal either by product formation. Purohit / Journal of Cleaner Production 11 (2003) 293–301 Fig. therefore. which are designed using the luciferase expression system and driven via specific promoters. These charac- teristics could only be provided. the disappearance of substrate. First. so that there is no delay in response time to release the signal. Various groups have taken the initiative to use this system and to utilize it as a monitoring tool in bioremediation. 3.

The resultant expression systems derived from these recombinant plasmids as biosensors. these kinds of biosensors have three main components. The chemistry of the reaction suggests that the same system could emit light of different wavelengths in different organisms [7]. The following are the promoters derived from various genes and which have been applied in environmental monitoring. The resultant complex is coupled with an oxidation of long chain aliphatic aldehyde such as n-decanal. which is proportional to expression of m-RNA or any reporter molecule. These are stress-induced proteins synthesized by some bacteria in conditions like nutrient starvation.J. The product of a reporter system has specific properties by itself. which is the sensing system at genetic level and the reporter system. In GFP. fischeri is reported to be stable up to 30 °C. In either of the situations. The gene product of luxAB is sufficient to generate the light signal if a long chain aliphatic aldehyde is directly provided in the reaction medium at concentration of 0. 4. either directly take the response of a target molecule or the target molecule interacts with promoter via a receptor system. Photorhadbus luminescens and others [8]. Promoters. V. Based on this property. Green fluorescent protein The green fluorescent protein (GFP) of the jellyfish Aequorea victoria absorbs light with an extinction maximum of 395 nm. The other two components are the promoter. GFP emits bright green light of wavelength 510 nm when excited with ultraviolet or blue light of wavelength 395 nm. or it catalyzes a biochemical reaction to generate the signal. the expression coded by Photorhadbus is reported to be stable up to 42 °C [10].1. which allows jellyfish to fluoresce due to transfer of energy from the Ca2+ activated photoprotein aequorin [7. The activity of this system could be directly monitored as light emitted. grpE or dnaK were sub-cloned in the lux expression vector.2. bacteria such as Pseudomonas could be used as an alternative host strain in monitoring the process of bioremediation of some compounds. luminescens is a terrestrial environment inhabitant. The series of reactions leading to emission of light are coded through the lux operon. Whereas. and others. 5. coli. Reporter systems The reporter system codes for a protein(s) and is a part of an expression vector. The promoters from E. in the application of bacterial bioluminescence. 5. 4.H. The lux operon (luxCDABE) has been cloned from Vibrio fischeri. An important distinction between them. the truncated expression vector with luxAB has been designed. particularly. which forms a hetrocyclic ring structure involving cyclization of Tyr66 amino acid residue [12]. To design a biological component for a biosensor. at the molecular level. Promoters as biosensors Promoters are 5 -flanking sequence in a gene or operon. exposure to toxic organics. and laboratory strains of Escherichia coli are the most conventional options. where the selection of promoter is based on the target molecule to be monitored in the samples.1. heavy metals. and fluoresces with emission maximum at 510 nm. which allows the maintenance and survival of biosensor in appropriate host. when challenged with different toxic . Thereby. fischeri is a marine bacterium and P. coli are not natural inhabitants of the soil. coli is used as the host.11]. is the variation in thermolability of their lux systems. The reaction uses the product of luxAB to code for luciferase activity.001%. It is an accessory protein in the bioluminescence pathway. the promoters are the actual sensing components of biosensors. which is generated by a fatty acid reductase complex and coded by luxCDE genes. The luciferase enzyme from V. a selected promoter sequence could be placed at the 5 -region of the reporter system. is generated via the tripeptide 65Ser-Tyr-Gly67. It has been applied in diverse ways with several target molecules such as xenobiotics or even oxygen in the medium [9]. which oxidizes reduced flavin mononucleotide in the presence of molecular oxygen to generate 4a-peroxyflavin. Purohit / Journal of Cleaner Production 11 (2003) 293–301 295 biosensor is the choice of the host. the response could be modulated kinetically. above which the enzyme starts losing its activity. have been observed to have a response time of less than 5 min. which could be received by a photomultiplier tube for signal analysis. which could be lux operon. 4. such as uspA. Generalized stress promoters Different kinds of environmental stress trigger groups include heat shock proteins. Thus. that respond to changes in cellular physiology and accordingly the genetic information stored in DNA is transcribed in terms of a messenger RNA. an internal chromophore that is responsible for the fluorescent property. The following are the most commonly deployed reporter systems. The vehicle or vector molecule (plasmid) is the principal component of this system. These promoters have been demonstrated for their non-specific response to various stresses when E. GFP or any other signal producing molecule. Since E. Bacterial luciferase reporter system Bioluminescence is an enzymatic response of luciferase activity.

Similarly. as these biosensors are nonspecific in their expression. The expression of this promoter decreases with decreased concentrations of DO level in the medium. The sensitivity of this system has been evaluated using different heterologous hosts. usage is possible only under defined conditions. However. The promoter based on oxidative stress KatG has been demonstrated for its application in monitoring DO level in the systems [24].3. then. Results have shown that under low pH conditions. Redox agents such as hydrogen peroxide.14]. The nitrogen starvation promoter glnA can be turned on with the nitrogen limiting conditions and phoA under the phosphorus limiting condition. and/or xylene in the contaminated sample [16]. where a bacterium that uses a pollutant as a carbon source could find the same pollutant as toxic above a particular concentration.J. and temperature. Considering this principle. The biosensors designed for mercury can target both Hg (II) present in the inorganic or organic form. in tropical countries with higher temperatures throughout the year. and for such monitoring the strains of E. The DNA molecule transferred in this type of experiment becomes lodged on the chromosome of the host DNA. the biosensor at the molecular level is flanked with the insertion sequences or sub-cloned in the transposon vector. organic peroxides. Biosensors that can be influenced by these parameters have also been reported.296 H. The effect of pH and toxicity of chlorophenols has been monitored by lux-based biosensor. where. the carbon source specific promoters can be applied. however. The plasmid based molecular biosensor multiplies in the host cells. The cells in the reactor were challenged by the stresses with different levels of target molecules and the expression of lux operon as the light signal. was recorded on-line [13. The biosensors can be designed for monitoring the level of nitrogen and phosphorus in the medium with an expression system using promoters. a lux reporter system derived from Photorhabdus could provide a more temperature stable expression system. polycyclic aromatic hydrocarbons [18]. Purohit / Journal of Cleaner Production 11 (2003) 293–301 molecules. which deals with the DNA damage. The levels of a pollutant in a medium is a critical parameter observed in the bioremediation program. coli has been observed to have a similar type of functioning. instead of using a plasmid as the vector the biosensors are introduced through the transposon system. In this scenario. toluene. For monitoring the bioavailabilty and water extractable mercury. A single-use Hg(II) biosensor comprised of immobilized cells has been designed which uses latex copolymer film and is able to detect with a sensitivity of 1 nM concentration [21]. This includes nutrient starvation and exposure to toxic chemicals [14]. influence the growth of bacteria. ethybenzene. The heat shock promoter grpE derived from E. coli were observed to be more sensitive hosts [23]. which has been shown to survive in the selected environmental conditions. The experiments were designed to monitor the signal through fiber optic probes. dicholorphenol is more toxic. characterized for detoxification of both the forms of Hg(II) [20]. The biosensor developed with this strategy has been demonstrated for monitoring of benzene. Temperature monitoring in bioremediation is not critical. Depending upon the conditions if survival of host bacterium or stability of the plasmid cannot be maintained. In this scenario. The metal ion specific biosensors are much explored in the area of mercury levels in the environmental sample. Monitoring of nutrients The bioremediation approach requires a typical C:N:P ratio for the success of biological degradation. since the lux operon derived from this bacterium is stable up to 42 °C. These independently developed biosensors use the specific promoters derived from mer operon. coli. This recombinant DNA is then transferred to a host. methyl viologen. 5. different bacterial strains were used as hosts with the lux reporter system to monitor the toxicity of phenol [17]. parameters like pH. Another promoter derived from the recA operon. respectively. the signal generation systems were designed. Monitoring of physical parameters Other than nutrient availability in the growth medium. and other redox cycling agents can also induce this promoter. has been . 5. and heavy metals [19]. a soil isolate-based biosensor has been designed. In case of Pseudomonas RB1351 the lux system driven by Pnah promoter has been expressed and it generates the signal in response to naphthalene in the medium. Monitoring of metal ion In the case of E. and was compared with commercially available toxicity monitoring kits. The studies were done in batch and continuous culture of these recombinant bacteria. This promoter is derived from the operon which is responsible for biodegradation of naphthalene [15]. This was engineered by transferring a mer-lux reporter system to a Pseudomonas putida strain [22]. 5. dissolved oxygen (DO).4. A monitoring tool for contaminated sites with petroleum products has been designed deriving the promoter from the tod operon. The promoter uspA derived from this gene can be switched on non-specifically by the conditions that limit cell growth. the universal stress protein A is encoded by uspA gene. biosensors that function at an optimum temperature of 30 °C would not be efficient. glnA and phoA. The generalized biosensors based on uspA and grpE have been demonstrated in the monitoring of heavy metals such as Cu2+ and Cd2+.2.

The signal generated is received through a fiber optic probe and correlated after digital processing for the level of toxicity. Therefore. To assess the toxicity of a wastewater stream. the xylR protein senses the hydrocarbon and after activation. With the immobilized cells. coli is a natural inhabitant of intestine. which uses the promoter xylS under the control of xylR. a two-stage mini bioreactor has been developed. But. how the toxicant will effect the host system. it has to be excited with a specific wavelength to produce the fluorescence phenomenon. This promoter could be induced by chemicals. The resultant sensor. The light signal generated in the luciferase system also can be received through the fiber optic device. In constructing a molecular biosensor the protocols use different genetic engineering steps. a bacterium from the soil or bacteria often observed in waste- . which harbors the molecular biosensor. However. In the luciferase expression system. which is converted into the light response. which are responsible for genotoxicity and even UV radiation exposure. Methodology The methodology of molecular biosensors can be discussed at two levels. Once a biosensor is developed. The multiple cloning sites are designed in a way that it does not interrupt the coding sequence for the reporter protein. The second reactor has physiologically consistent cells. the protein is synthesized and to generate the signal. which in turn would modulate the generation of the signal. The construction of the recombinant plasmid used in this article shows that the target promoter was amplified using the polymerase chain reaction [7. which is fixed at the end of fiber optic probe. The generalized scheme for a recombinant plasmid as a molecular biosensor has a configuration like any other expression vector. the exhaustion of substrate or generation of product is correlated with the signal. The signal is generated from the stimulation received by the promoter. In one reactor. Hence. Prior knowledge of the gene or genetic operon is the starting point of conceptualizing any biosensor. which is connected to a data processing unit to digitize the signal. reacts with the promoter to turn on the lux reporter gene which is used for generation of the light signal [29]. species-independent and can be monitored noninvasively using the technique of fluorescence microscopy and flow cytometry. Purohit / Journal of Cleaner Production 11 (2003) 293–301 297 extensively explored for its application in biosensors. the designing of the biosensor and secondly. Thus. The signal is generated as a response to target molecule. Examples of on-line monitoring The on-line monitoring using lux as a reporter system in biosensors has been demonstrated by various groups. generates the light signal mediated through the lux reporter system.26]. 6. a regulatory protein. The interaction of the target molecule to recombinant bacteria is essentially addressed in the aforementioned monitoring tools. The quantification of the generated signal is the most crucial step.H. chemicals such as benzene in the vapor state have been shown to induce the light response on-line. which is crucial for sensitivity of the response [27].11. under stress conditions i. in its application as a monitoring tool where the appropriate signal generation options are utilized. the luminescence signal could be quenched on the photographic film or by using a luminometer to quantify the signal. and its level could be determined as a function of target molecule and promoter interaction. However. the biosensor strain is kept in a continuous cultivation mode with the desired dilution rate. the promoter of this gene could be considered as a candidate for developing the biosensor. The bacterial cells are either used as immobilized cells or in suspended growth culture. In case of GFP. In the later type. First. The signal captured on the photographic plate can be analyzed by the densitometric analysis. coli as a host.J. GFP fluorescence is stable. It has a reporter system with multiple cloning sites at the 5 -end to sub-clone the promoter.e. the selected physiological response could be assigned to a key gene or operon. A typical physiological response is due to expression of a series of genetic events in the cells. The property of this promoter has been extended to evaluate the relative quotients for genotoxicant molecule [25. [28]. The signal could either be directly a protein or something that acts as a functional protein to bring out the biochemical reaction.15]. on exposure to gases carrying hydrocarbons. then capturing the generated signal and its quantification is the second step. Since E. At the molecular level. The immobilization is carried out in agar medium layered in a polypropylene tube. it has been shown in suspended growth cultivation either in the batch or continuous mode. A more specific promoter of a similar nature designed to monitor benzene and its derivatives has been reported. that it can be processed quantitatively. that it is the growth phase. there could be a gene or an operon that plays a crucial role in the manifestation of that physiological response. which are stimulated by a wastewater stream or challenged with a toxic molecule. all these studies used E. the second reactor behaves as a reaction vessel for light signal quantification [30]. in this sequence of reactions. which gives the cells a physiology having maximum sensing capacity. exposure to a toxicant. has not been thoroughly studied. so 7. However. The primers used in the amplification provide the restriction digestion sites for directional sub-cloning. to target and monitor this physiological response under different conditions.

which has a lot of potential for the different low-cost monitoring devices. This requires designing of a plasmid having a broad host range localization and survival capacity. Therefore. The different designs of mini bioreactors and associated growth kinetics. To design these strategies. since the host strain for a biosensor remains under controlled conditions in a specially devised contactor. the signal processing should be more cost-effective. there is always an expected DO level in the treated effluent. The role of different proteins as biosensors is a growing area of research. Fig. instead of using the conventional reporter systems such as lux or GFP. the control microcosm experiments of short duration at lab scale having a GFP as a reporter system.298 H. research is needed in the selection of appropriate hosts. a functional protein such as an enzyme can be used as the reporter system. The enzyme can react directly with the toxicant molecule to generate a product. then the cumulative light signal could be directly processed and displayed through LCD. In case of the promoter-based biosensors. as an option. In the case where recombinant plasmids are used. for an off-the-shelf product efforts are required to design a suitable biosensor holding assembly. where the microbial community structures play a key role in pollutant removal [33]. there must be provision for simultaneous monitoring of DO level in the effluent. The future of biosensors Molecular biosensors provide the specificity and sensitivity. The fluorophore molecule. The host cells harbor the genetic information to generate signal through reporter system as lux or GFP to monitor the level of pollutant. host and recombinant plasmid. it does not pose any threat of releasing its recombinant DNA. The contactor reactor. and digitization of this signal are the research areas in most urgent need. 8. However. the contactor assembly could be left at room temperature and the signal could be monitored. The host cells and growth medium could be hydrated using wastewater with the pollutants to be analyzed. The genetic variants of both the systems have been thoroughly tested at the laboratory scale. since it could allow the in situ monitoring of developing microbial communities. The DO level in the effluent is an indirect measure of associated biological activity in the treatment plant. could be useful. Most of the reported contactors (reaction module for host–target molecule interaction) use direct reactions of toxicant with host cells. where the biological component interacts with the target molecule and generates the signal. This area of research essentially targets the designing of innovative reporter systems. have to be tailormade for the pollutant specific remediation strategy. one-time use biosensors or off-line biosensors are another area for research. could be a choice of host for these types of studies. The future research for application of biosensors in bioremediation should be focused upon different levels of development. First. However. The biological components. The lux expression system also needs a continuous cellular pool of long chain fatty acids as a substrate. where they have been covalently linked directly or conjugated with chromophore groups and applied in different analysis [31]. the host cells carrying biosensors could be lyophilized cells with supporting nutrients in a desired physiological condition. . which can act as a signal generating species or it can be coupled with a fluorophore molecule that generates the light signal. where the cellular physiology is not over-burdened due to the additional task of synthesizing new proteins coded by the reporter system. The scheme suggests that before the effluent is discharged. require more application-friendly tools as shown in Fig. the developed biosensor could be mobilized in a naturally occurring bacterium from a given habitat so that its survival is assured. In accidental contamination where the target sites are not ready for assimilation of pollutants the bioaugmentation strategies have to be devised to decontaminate the sites. Findings on the role of fluorophore molecules in signal transmission and enhancement could support development of this technology [32]. the level of pollutant is assessed based on the host cells with biosensor monitoring system in the interfacing sac where it will respond at the cost of the level of pollutant. is still not a thoroughly explored area. In this case. including the GFP could support this type of study. which is analogous to the contactor proposed for on-line monitoring. Moreover. Depending upon the cell response time. The frequently used reporter systems. however. In either of the cases. Purohit / Journal of Cleaner Production 11 (2003) 293–301 water treatment plants. The reactor. Bioremediation is an emerging area of research in waste management. GFP has been demonstrated in horizontal transfer of catabolic plasmids amongst the acclimatizing microbial population [34]. coli are not robust enough as a host to harbor a molecular sensor to be applied in a bioremediation program. 2 suggests two types of observations. where the target molecule and the recipi- ent host strain carrying biosensor interact. 2. The usage of such systems at lab scale is easily demonstrable. In generating real time biosensors. their safe disposal becomes an obligation to the user. the laboratory strains of E. which are essential features that a monitoring tool demands. The different fluorescent proteins. luciferase and GFP. the supplementary data on DO level provide an internal check for residual level of the pollutant in the effluent. Secondly. This kind of plasmid can carry the molecular biosensor and could be mobilized within a more robust strain. provide reproducible results. For an efficiently running treatment plant. Alternatively. If lux is used as the reporter system.J. could be involved as a signal amplification system.

finally. 2. . Based on the DO data and if the lux expression is also high. could have case-specific applications. and fed with growth medium where the kinetics has been worked out. In monitoring the efficiency of treatment of wastewater with the expression system. which could be as follows. it will turn off the organic loading till it stabilizes. the biosensors based on gross biological capacities have already been reported [35]. it will switch on the pump to inject phosphorus source in the plant and wait. which is based on the probe studies [37]. Schematic diagram for maintaining treatment efficiency of a biological wastewater treatment system. As observed by the other workers in this area of research. In wastewater treatment processes. The proposed system relies on parallel analysis. the biosensors described in Section 7. The decision will be based on knowledge provided to the controller via both the types of analyses as proposed in Fig. Biological signal generating system comprises mini bioreactor having (for example) generalized stress promoter usp::luxCDABE for monitoring of pollutant. as described earlier. then. The flow in both the tubes is in the same direction and allows the target molecule to pass through the membrane to generate the signal. then. This promoter drives a truncated lux reporter system to monitor phenol levels. these types of systems can be used to generate knowledge for a specific effluent and expected shocks the treatment system could face. We are working on a system where the promoter has been derived from the gene. However. The process will provide parameter based continuous treatment as a function of biosensor output. it will follow the same loop.H. If systems consider still a problem then. The gradation in the tube shows the response from the cells. The development and applications of the developed biosensors is still at laboratory trial stage. The recombinant cells with a desired dilution rate in continuous mode enter in an interfacing contactor. The generated knowledge with trouble shooting options. Interfacing contactor has been proposed with an interfacing sac (membrane with required cut off) through which the biosensor culture passes. the contactor where the biosensor would react with target molecule is the rate-limiting step in transfer of technology in the field. Initially.J. where microorganisms use the pollutants from the effluent as a food for survival. These types of biosensors use whole cell preparations to monitor parameters such as DO level [20]. could be fed to the control panel of the treatment plant. which will have in-built knowledge to maintain the treatment efficiency. a case-specific decision-making tool can be envisaged as shown in Fig. Purohit / Journal of Cleaner Production 11 (2003) 293–301 299 Fig. If there is still high DO and lux expression. whereas the outer part has another concentric tube through which the wastewater could flow. The light signal and DO levels will provide the in-put data for controller unit. The waste after passing through the interfacing contactor goes via flow-through cells having DO measurement. which is responsible for phenol utilization. controller will first inject nitrogen source in the treatment plant and wait for the results. 2. 2. We are also extending the same system to where it can monitor the availability of NH4 as nitrogen source and its relationship to phenol level [36]. accordingly a sequential algorithm could be followed with the in-built options for interim corrections.

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