Journal of Cleaner Production 11 (2003) 293301 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 specicity 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 scientic 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 eld 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 specicity is used as the basis in designing biosensors. Therefore, a biosensor can be case-specic in its approach or with application of genetic engineering; more exible biosensors can be also envisaged. The second component of this analytical device is a signalgenerating surface, which feeds the information ow to a signal-processing unit. There are different schematic diagrams available in the literature describing biosensors [1,2]. However, a biosensor is mostly case-specic

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 exible biosensors. This gure 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 specic 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].

Fax: +91-712-222725. E-mail address: hemantdrd@hotmail.com (H.J. Purohit).

0959-6526/02/$ - see front matter. 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 9 - 6 5 2 6 ( 0 2 ) 0 0 0 7 2 - 0

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Fig. 1. Schematic depiction of target molecule is captured by ligand

interacting with the biological component to yield signal molecule, which

to pass on the signal to processing unit to generate electronic output.

There could be another option where the inherent property of a cell or tissue is used to generate the signal [4]. Of the different types of biosensors, the enzymebased biosensors have been extensively explored. Biosensors using enzymes in their biological function generate the signal either by product formation, the disappearance of substrate, or co-enzyme conversion. Sometimes these reactions are superimposed with another biochemical event such as use of inhibition kinetics or coupling with other reaction(s). Various enzymebased biosensors are reported for the detection of pesticides in different samples. The enzymes most often applied are cholinesterase and choline oxidase for the detection of organophosphorous pesticides [5]. Recent advances in clinical sample analysis show a trend where biosensors based on luminescence systems are used [6]. Various groups have taken the initiative to use this system and to utilize it as a monitoring tool in bioremediation. Bioremediation is an application of microbial capacity to transform complex organic molecules into simpler inorganic constituents. Therefore, it represents an environment where the microbial community is thriving as they biodegrade various pollutants. In such an environment, biosensors with the following basic features would be suitable. First, it should not affect the microbial community structure if it is to be used as whole cells. Secondly, the specicity of monitoring towards the target molecule should be independent of changes in environmental conditions. These charac-

teristics could only be provided, if the biological component through the rest of the environment, is separated; but still is kept sufciently interactive, so that there is no delay in response time to release the signal. In this article the author describes such biosensors, which are designed using the luciferase expression system and driven via specic promoters. They are described as molecular biosensors since they use the promoters derived from specic genes to interact with target molecules.

3. Molecular biosensors The biological component in a molecular biosensor is a recombinant plasmid. It has a specic promoter, whose expression is sensitive to a target molecule and uses the reporter system to generate the signal. The promoters can be turned on or off with specic molecules, hence they provide the required specicity in signal generation. The generation of signals is directly proportional to the expression of the promoter. The case-specic recombinant plasmids can be genetically engineered so as to lead to a biosensor. Sometimes, it is divided in a manner that the host chromosome carries a part of the supporting activity for expression of the promoter. For this type of application, the additional requirement is a support system for the survival and multiplication of these designed sensing molecules. Another feature, therefore, in such a

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biosensor is the choice of the host; and laboratory strains of Escherichia coli are the most conventional options. Since E. coli are not natural inhabitants of the soil, bacteria such as Pseudomonas could be used as an alternative host strain in monitoring the process of bioremediation of some compounds.

4. Reporter systems The reporter system codes for a protein(s) and is a part of an expression vector. The product of a reporter system has specic properties by itself, or it catalyzes a biochemical reaction to generate the signal. The following are the most commonly deployed reporter systems. 4.1. Bacterial luciferase reporter system Bioluminescence is an enzymatic response of luciferase activity. The chemistry of the reaction suggests that the same system could emit light of different wavelengths in different organisms [7]. The series of reactions leading to emission of light are coded through the lux operon. The lux operon (luxCDABE) has been cloned from Vibrio scheri, Photorhadbus luminescens and others [8]. The reaction uses the product of luxAB to code for luciferase activity, which oxidizes reduced avin mononucleotide in the presence of molecular oxygen to generate 4a-peroxyavin. The resultant complex is coupled with an oxidation of long chain aliphatic aldehyde such as n-decanal, which is generated by a fatty acid reductase complex and coded by luxCDE genes. The gene product of luxAB is sufcient to generate the light signal if a long chain aliphatic aldehyde is directly provided in the reaction medium at concentration of 0.001%. Based on this property, the truncated expression vector with luxAB has been designed. It has been applied in diverse ways with several target molecules such as xenobiotics or even oxygen in the medium [9]. V. scheri is a marine bacterium and P. luminescens is a terrestrial environment inhabitant. An important distinction between them, particularly, in the application of bacterial bioluminescence, is the variation in thermolability of their lux systems. The luciferase enzyme from V. scheri is reported to be stable up to 30 C, above which the enzyme starts losing its activity. Whereas, the expression coded by Photorhadbus is reported to be stable up to 42 C [10]. The activity of this system could be directly monitored as light emitted, which could be received by a photomultiplier tube for signal analysis. 4.2. Green uorescent protein The green uorescent protein (GFP) of the jellysh Aequorea victoria absorbs light with an extinction

maximum of 395 nm, and uoresces with emission maximum at 510 nm. It is an accessory protein in the bioluminescence pathway, which allows jellysh to uoresce due to transfer of energy from the Ca2+ activated photoprotein aequorin [7,11]. In GFP, an internal chromophore that is responsible for the uorescent property, is generated via the tripeptide 65Ser-Tyr-Gly67, which forms a hetrocyclic ring structure involving cyclization of Tyr66 amino acid residue [12]. GFP emits bright green light of wavelength 510 nm when excited with ultraviolet or blue light of wavelength 395 nm.

5. Promoters as biosensors Promoters are 5 -anking sequence in a gene or operon, that respond to changes in cellular physiology and accordingly the genetic information stored in DNA is transcribed in terms of a messenger RNA. Promoters, either directly take the response of a target molecule or the target molecule interacts with promoter via a receptor system. In either of the situations, the response could be modulated kinetically, which is proportional to expression of m-RNA or any reporter molecule. To design a biological component for a biosensor, a selected promoter sequence could be placed at the 5 -region of the reporter system, where the selection of promoter is based on the target molecule to be monitored in the samples. Thereby, at the molecular level, the promoters are the actual sensing components of biosensors. Thus, these kinds of biosensors have three main components. The vehicle or vector molecule (plasmid) is the principal component of this system, which allows the maintenance and survival of biosensor in appropriate host. The other two components are the promoter, which is the sensing system at genetic level and the reporter system, which could be lux operon, GFP or any other signal producing molecule. The following are the promoters derived from various genes and which have been applied in environmental monitoring. 5.1. Generalized stress promoters Different kinds of environmental stress trigger groups include heat shock proteins. These are stress-induced proteins synthesized by some bacteria in conditions like nutrient starvation, exposure to toxic organics, heavy metals, and others. The promoters from E. coli, such as uspA, grpE or dnaK were sub-cloned in the lux expression vector. These promoters have been demonstrated for their non-specic response to various stresses when E. coli is used as the host. The resultant expression systems derived from these recombinant plasmids as biosensors, have been observed to have a response time of less than 5 min, when challenged with different toxic

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molecules. The studies were done in batch and continuous culture of these recombinant bacteria. The experiments were designed to monitor the signal through ber optic probes. 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 [13,14]. 5.2. Monitoring of nutrients The bioremediation approach requires a typical C:N:P ratio for the success of biological degradation. The biosensors can be designed for monitoring the level of nitrogen and phosphorus in the medium with an expression system using promoters, glnA and phoA, respectively. The nitrogen starvation promoter glnA can be turned on with the nitrogen limiting conditions and phoA under the phosphorus limiting condition. Similarly, the carbon source specic promoters can be applied. 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. This promoter is derived from the operon which is responsible for biodegradation of naphthalene [15]. A monitoring tool for contaminated sites with petroleum products has been designed deriving the promoter from the tod operon. The plasmid based molecular biosensor multiplies in the host cells. Depending upon the conditions if survival of host bacterium or stability of the plasmid cannot be maintained, then, instead of using a plasmid as the vector the biosensors are introduced through the transposon system. In this scenario, the biosensor at the molecular level is anked with the insertion sequences or sub-cloned in the transposon vector. This recombinant DNA is then transferred to a host, which has been shown to survive in the selected environmental conditions. The DNA molecule transferred in this type of experiment becomes lodged on the chromosome of the host DNA. The biosensor developed with this strategy has been demonstrated for monitoring of benzene, toluene, ethybenzene, and/or xylene in the contaminated sample [16]. The levels of a pollutant in a medium is a critical parameter observed in the bioremediation program; where a bacterium that uses a pollutant as a carbon source could nd the same pollutant as toxic above a particular concentration. Considering this principle, the signal generation systems were designed; where, different bacterial strains were used as hosts with the lux reporter system to monitor the toxicity of phenol [17], polycyclic aromatic hydrocarbons [18], and heavy metals [19]. 5.3. Monitoring of metal ion In the case of E. coli, the universal stress protein A is encoded by uspA gene. The promoter uspA derived

from this gene can be switched on non-specically by the conditions that limit cell growth. This includes nutrient starvation and exposure to toxic chemicals [14]. The heat shock promoter grpE derived from E. coli has been observed to have a similar type of functioning. The generalized biosensors based on uspA and grpE have been demonstrated in the monitoring of heavy metals such as Cu2+ and Cd2+. However, as these biosensors are nonspecic in their expression, usage is possible only under dened conditions. The metal ion specic biosensors are much explored in the area of mercury levels in the environmental sample. The biosensors designed for mercury can target both Hg (II) present in the inorganic or organic form. These independently developed biosensors use the specic promoters derived from mer operon, characterized for detoxication of both the forms of Hg(II) [20]. A single-use Hg(II) biosensor comprised of immobilized cells has been designed which uses latex copolymer lm and is able to detect with a sensitivity of 1 nM concentration [21]. For monitoring the bioavailabilty and water extractable mercury, a soil isolate-based biosensor has been designed. This was engineered by transferring a mer-lux reporter system to a Pseudomonas putida strain [22]. 5.4. Monitoring of physical parameters Other than nutrient availability in the growth medium, parameters like pH, dissolved oxygen (DO), and temperature, inuence the growth of bacteria. Biosensors that can be inuenced by these parameters have also been reported. The effect of pH and toxicity of chlorophenols has been monitored by lux-based biosensor. The sensitivity of this system has been evaluated using different heterologous hosts; and was compared with commercially available toxicity monitoring kits. Results have shown that under low pH conditions, dicholorphenol is more toxic; and for such monitoring the strains of E. coli were observed to be more sensitive hosts [23]. The promoter based on oxidative stress KatG has been demonstrated for its application in monitoring DO level in the systems [24]. The expression of this promoter decreases with decreased concentrations of DO level in the medium. Redox agents such as hydrogen peroxide, methyl viologen, organic peroxides, and other redox cycling agents can also induce this promoter. Temperature monitoring in bioremediation is not critical; however, in tropical countries with higher temperatures throughout the year, biosensors that function at an optimum temperature of 30 C would not be efcient. In this scenario, a lux reporter system derived from Photorhabdus could provide a more temperature stable expression system, since the lux operon derived from this bacterium is stable up to 42 C. Another promoter derived from the recA operon, which deals with the DNA damage, has been

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extensively explored for its application in biosensors. This promoter could be induced by chemicals, which are responsible for genotoxicity and even UV radiation exposure. The property of this promoter has been extended to evaluate the relative quotients for genotoxicant molecule [25,26].

that it can be processed quantitatively. In case of GFP, the protein is synthesized and to generate the signal, it has to be excited with a specic wavelength to produce the uorescence phenomenon. GFP uorescence is stable, species-independent and can be monitored noninvasively using the technique of uorescence microscopy and ow cytometry.

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

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water treatment plants, could be a choice of host for these types of studies.

8. The future of biosensors Molecular biosensors provide the specicity and sensitivity, which are essential features that a monitoring tool demands. The future research for application of biosensors in bioremediation should be focused upon different levels of development. The biological components, host and recombinant plasmid, have to be tailormade for the pollutant specic remediation strategy. The frequently used reporter systems, luciferase and GFP, provide reproducible results. The genetic variants of both the systems have been thoroughly tested at the laboratory scale. The contactor reactor, where the biological component interacts with the target molecule and generates the signal, and digitization of this signal are the research areas in most urgent need. In generating real time biosensors, research is needed in the selection of appropriate hosts, where the cellular physiology is not over-burdened due to the additional task of synthesizing new proteins coded by the reporter system. The lux expression system also needs a continuous cellular pool of long chain fatty acids as a substrate. Moreover, the laboratory strains of E. coli are not robust enough as a host to harbor a molecular sensor to be applied in a bioremediation program. This requires designing of a plasmid having a broad host range localization and survival capacity. This kind of plasmid can carry the molecular biosensor and could be mobilized within a more robust strain. Alternatively, the developed biosensor could be mobilized in a naturally occurring bacterium from a given habitat so that its survival is assured. In either of the cases, since the host strain for a biosensor remains under controlled conditions in a specially devised contactor, it does not pose any threat of releasing its recombinant DNA. The role of different proteins as biosensors is a growing area of research, where they have been covalently linked directly or conjugated with chromophore groups and applied in different analysis [31]. Findings on the role of uorophore molecules in signal transmission and enhancement could support development of this technology [32]. This area of research essentially targets the designing of innovative reporter systems. In this case, instead of using the conventional reporter systems such as lux or GFP, 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, which can act as a signal generating species or it can be coupled with a uorophore molecule that generates the light signal. The uorophore molecule, as an option, could be involved as a signal amplication system. The reactor, where the target molecule and the recipi-

ent host strain carrying biosensor interact, is still not a thoroughly explored area. The different designs of mini bioreactors and associated growth kinetics, require more application-friendly tools as shown in Fig. 2. Most of the reported contactors (reaction module for hosttarget molecule interaction) use direct reactions of toxicant with host cells. The host cells harbor the genetic information to generate signal through reporter system as lux or GFP to monitor the level of pollutant. However, Fig. 2 suggests two types of observations. First, 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. The scheme suggests that before the efuent is discharged, there must be provision for simultaneous monitoring of DO level in the efuent. The DO level in the efuent is an indirect measure of associated biological activity in the treatment plant. For an efciently running treatment plant, there is always an expected DO level in the treated efuent. Therefore, the supplementary data on DO level provide an internal check for residual level of the pollutant in the efuent. Secondly, one-time use biosensors or off-line biosensors are another area for research, which has a lot of potential for the different low-cost monitoring devices. In the case where recombinant plasmids are used, their safe disposal becomes an obligation to the user. In case of the promoter-based biosensors, the host cells carrying biosensors could be lyophilized cells with supporting nutrients in a desired physiological condition. The usage of such systems at lab scale is easily demonstrable. However, for an off-the-shelf product efforts are required to design a suitable biosensor holding assembly, which is analogous to the contactor proposed for on-line monitoring. The host cells and growth medium could be hydrated using wastewater with the pollutants to be analyzed. Depending upon the cell response time, the contactor assembly could be left at room temperature and the signal could be monitored. If lux is used as the reporter system, then the cumulative light signal could be directly processed and displayed through LCD. Bioremediation is an emerging area of research in waste management, where the microbial community structures play a key role in pollutant removal [33]. The different uorescent proteins, including the GFP could support this type of study, however, the signal processing should be more cost-effective. GFP has been demonstrated in horizontal transfer of catabolic plasmids amongst the acclimatizing microbial population [34]. 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. To design these strategies, the control microcosm experiments of short duration at lab scale having a GFP as a reporter system, could be useful, since it could allow the in situ monitoring of developing microbial communities.

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Fig. 2. Schematic diagram for maintaining treatment efciency of a biological wastewater treatment system. Biological signal generating system comprises mini bioreactor having (for example) generalized stress promoter usp::luxCDABE for monitoring of pollutant; and fed with growth medium where the kinetics has been worked out. The recombinant cells with a desired dilution rate in continuous mode enter in an interfacing contactor. Interfacing contactor has been proposed with an interfacing sac (membrane with required cut off) through which the biosensor culture passes, whereas the outer part has another concentric tube through which the wastewater could ow. The ow in both the tubes is in the same direction and allows the target molecule to pass through the membrane to generate the signal. The gradation in the tube shows the response from the cells. The waste after passing through the interfacing contactor goes via ow-through cells having DO measurement. The light signal and DO levels will provide the in-put data for controller unit, which will have in-built knowledge to maintain the treatment efciency, which could be as follows. Based on the DO data and if the lux expression is also high, then, controller will rst inject nitrogen source in the treatment plant and wait for the results. If there is still high DO and lux expression, then, it will switch on the pump to inject phosphorus source in the plant and wait. If systems consider still a problem then, it will turn off the organic loading till it stabilizes; nally, it will follow the same loop.

In wastewater treatment processes, the biosensors based on gross biological capacities have already been reported [35]. These types of biosensors use whole cell preparations to monitor parameters such as DO level [20]. However, the biosensors described in Section 7, could have case-specic applications. We are working on a system where the promoter has been derived from the gene, which is responsible for phenol utilization. This promoter drives a truncated lux reporter system to monitor phenol levels. 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]. The development and applications of the developed biosensors is still at laboratory trial stage. As observed by the other workers in this area of research, the contactor where the biosensor would react with target molecule is the rate-limiting step in transfer of technology in the eld.

In monitoring the efciency of treatment of wastewater with the expression system, a case-specic decision-making tool can be envisaged as shown in Fig. 2, which is based on the probe studies [37]. The proposed system relies on parallel analysis, as described earlier. Initially, these types of systems can be used to generate knowledge for a specic efuent and expected shocks the treatment system could face, where microorganisms use the pollutants from the efuent as a food for survival. The generated knowledge with trouble shooting options, could be fed to the control panel of the treatment plant. The decision will be based on knowledge provided to the controller via both the types of analyses as proposed in Fig. 2; accordingly a sequential algorithm could be followed with the in-built options for interim corrections. The process will provide parameter based continuous treatment as a function of biosensor output.

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To summarize, biosensors, the technology of the future, may increasingly rely on the structure and function-specicity of the biological component. Assuring the non-invasive interfacing with the target molecule through mini reactors will provide the next generation of signal generating systems. The signal directly or supported through the uorophore molecules, will help in decision-making in technologies based on biological systems. References
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