Microbial Biosensor and Bioelectronics

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					Research Journal of Biotechnology                                                                            Vol. 7 (3) August (2012)
                                                                                                                        Res. J. Biotech

Review paper:
                  Microbial Biosensors and Bioelectronics
                                         Somayeh Dolatabadi* and Manjulakumari D.
                         Dept. of Microbiology and Biotechnology, Bangalore University, Bangalore-560056, INDIA

Abstract                                                                 multipurpose catalyst especially when the process requires
A microbial biosensor consists of a transducer in                        the participation of a number of enzymes in sequence.
conjunction with immobilized viable or non-viable
microbial cells. Non-viable cells obtained after                         Another mechanism used for the viable microbial biosensor
                                                                         involves the inhibition of microbial respiration by the
permeabilization or whole cells containing peri-
                                                                         analyte of interest, like environmental pollutants. The
plasmic enzymes have mostly been used as an                              major limitation to the use of whole cells is the diffusion of
economical substitute for enzymes. Viable cells make                     substrate and products through the cell wall resulting in a
use of the respiratory and metabolic functions of the                    slow response as compared to enzyme- based sensors. The
cell, while the analyte to be monitored is either a                      cells can be permeabilised using physical (freezing and
substrate or an inhibitor of these processes.                            thawing), chemical (organic solvents/detergents) and
Bioluminescence-based microbial biosensors have                          enzymatic (lysozyme, papain) approaches. The most
also been developed using genetically engineered                         common technique uses organic solvents. Such chemical
microorganisms constructed by fusing the lux gene                        treatment creates minute pores by removing some of the
with an inducible gene promoter for toxicity and                         lipids from the cell membranes, thereby allowing for the
                                                                         free diffusion of small molecular weight substrates/
bioavailability testing.
                                                                         products across the cell membrane.
Keywords: Microbial biosensors, Immobilized cells,
                                                                         These types of approaches may have major significance in
Bioluminescence biosensors, Permeabilised cells, Environ-
                                                                         the future, especially for sensors like BOD where in
mental biosensors.
                                                                         polymers such as protein, starch, lipid etc have to be broken
                                                                         down to monomers before they can be metabolized.
Introduction                                                             Recently, genetically engineered cells have been obtained
A biosensor is a device that detects, transmits and records              for expression of cellulase activity on the cell surface. Such
information regarding a physiological or biochemical                     modified whole cells have been shown to hydrolyze the
change. The choice of the biological material as a sensing               cellulose from the media and can replace the use of acid-
material will depend on a number of factors like the                     induced breakdown of biological polymers prior to
specificity, storage, operational and environmental stability.           biosensor analysis.
Selection also depends on the analyte to be detected such as
chemical compounds, antigens, microbes, hormones,                        Another limitation in using whole cells is the low
nucleic acids or any subjective parameters like smell and                specificity as compared to biosensors containing pure
taste. Enzymes, antibodies, DNA, receptors, organelles and               enzymes. Several approaches are being investigated to
microorganisms as well as animal and plant cells or tissues              minimize such non-specific reactions. Permeabilisation of
have been used as biological sensing elements in a wide                  the cell empties most of the small molecular weight
range of fields.                                                         cofactors etc, thus minimizing the unwanted side reactions.
                                                                         Another approach that is of significance in viable cell-
Use of microbial cells as biosensor elements                             based biosensors is the blockage of unwanted metabolic
Microbes have a number of advantages as biological                       pathways or transport systems.
sensing materials in the fabrication of biosensors. They are
present ubiquitously and are able to metabolize a wide                   Developments in recombinant DNA technology may help
range of chemical compounds. Microorganisms have a                       in producing whole cells rich in the enzyme of interest and
great capacity to adapt to adverse conditions and to develop             also engineered to have minimal amounts of enzymes that
the ability to degrade new molecules with time. In this                  might catalyze side reactions. Adaptation of a microbe for
respect, the utilization of whole cells as a source of                   induction of desirable metabolic pathways and uptake
intracellular enzymes has been shown to be a better                      systems by cultivation in medium containing appropriate
alternative to purified enzymes in various industrial                    substrates may often be desirable.
                                                                         Microbial biosensors based on light emission from
It avoids the lengthy and expensive operations of enzyme                 luminescent bacteria are being applied as a sensitive, rapid
purification, preserves the enzyme in its natural                        and non-invasive assay in several biological systems.
environment and protects it from inactivation by external                Bioluminescent bacteria are found in nature, their habitat
toxicants such as heavy metals. Whole cells also provide a               ranging from marine (Vibrio fischeri) to terrestrial
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(Photorhabdus luminescens) environments. Bioluminescent                  sensor was not influenced by NaCl even up to 30% (w/v).
whole cell biosensors have also been developed using                     The first commercial BOD sensor was produced by the
genetically engineered microorganisms (GEM) for the                      Japanese company Nisshin Electric in 1983 and a number
monitoring of organic, pesticide and heavy metal                         of other commercial BOD biosensors based on viable
contamination. The microorganisms used in these                          microbial cells are being marketed. Another BOD sensor
biosensors are typically produced with a constructed                     based on a soil bacterium Pseudomonas putida capable of
plasmid in which genes that code for luciferase are placed               determining low BOD levels in river water and secondary
under the control of a promoter that recognizes the analyte              effluents and exhibiting negligible response to interference
of interest. When such microbes metabolize the organic                   by chloride and heavy metals has been reported.
pollutants, the genetic control mechanism also turns on the
synthesis of luciferase, which produces light that can be                The instrument is commercially available through Central
detected by luminometers.                                                Kagaku Corp., Tokyo. The use of these devices has been
                                                                         incorporated into industrial standard methods in Japan13.
A useful reporter system responsible for light emission is               Significant efforts have been made towards the
made up of five structural genes, luxCDABE, of the                       development of a portable BOD biosensor system
bioluminescence operon derived from marine bacterium V.                  incorporating disposable electrodes. Miniature Clark-type
fischeri. The luxCDE genes encode an enzyme complex                      oxygen electrode arrays were fabricated using thin film
(fatty acid reductase, synthetase and transferase) that                  technology for mass production with assured quality. A
synthesizes the substrate (a fatty aldehyde) for luciferase,             microbial biosensor consisting of an oxygen microelectrode
using precursors from the fatty acid cycle. The luxAB                    with microbial cells immobilized in polyvinyl alcohol has
genes encode the luciferase enzyme16.                                    been fabricated for the measurement of bioavailability of
                                                                         organic carbon. The biosensor allows the estimation of
Bacterial strains that increase light production in the                  available dissolved organic carbon in sediment profiles on a
presence of specific chemicals have been constructed using               micro scale. Optical fiber and calorimetric based
bioluminescence genes (lux) as reporters of transcriptional              transducers have been used in BOD biosensors.
responses. A complementary approach, not requiring prior
knowledge of expected contaminants, uses less specific                   Microbial biosensors have been investigated for a variety of
stress responses as general indicators of deleterious                    other environmental applications e.g. in detection of
conditions. Cellular organelles can be considered to be                  environmental pollutants. Halogenated hydrocarbons used
multifunctional biocatalysts, intermediate in complexity                 as pesticides, foaming agents, flame-retardants,
between whole cells and enzymes.                                         pharmaceuticals and intermediates in the polymer
                                                                         production are one of the largest group of environmental
Microbial biosensors for environmental applica-                          pollutants. The enzyme present in the cell liberates halogen
tions                                                                    ions from halogenated hydrocarbons. These studies were
The major application of microbial biosensors is in the                  extended in the fabrication of a microbial sensor. The
environmental field. Microbial biosensors have been                      sensor can be stored in the dry form at 277 K for 1 week.
developed for assaying BOD, a value related to total                     Major disadvantage was the additional pre- incubation
content of organic materials in wastewater. BOD sensors                  period of 30 min that is required before the electrode
take advantage of the high reaction rates of microorganisms              potential attains stability.
interfaced to electrodes to measure the oxygen depletion
rates. Standard BOD assay requires 5 days compared to 15                 More recently a gram-positive actinomycete-like organism,
min for a biosensor-based analysis. Ever since the first                 exhibiting a broad spectrum for the dehalogenation of
report of such a microbial sensor1 was made, a large                     halogenated hydrocarbons, has shown better promise and
number of papers have appeared in this field and is                      may have potential in the fabrication of a broad specificity
perhaps, the most extensively investigated microbial                     biosensor for halogenated hydrocarbons. The strain of
biosensor. One of the major criteria in the selection of these           Xanthobacter autotrophicus GJ 10, a nitrogen-fixing
microbes is that they should be able to utilize a very broad             microbe can utilize 1,2 dichloroethane as the sole source of
range of substrates as discussed above. The biosensor                    carbon and energy. The bacterium is known to form two
should also be stable to environmental adversaries such as               halogenases. One is specific for the dehalogenation of
heavy metal toxicity, salinity etc.                                      halogenated alkanes; other converts halogenated carboxylic
                                                                         acids by hydrolytic cleavage into corresponding alcohol
Recent studies on a salt tolerant dimorphic (budding and                 and halogen ion. Polycyclic aromatic hydrocarbons (PAH)
mycelial) yeast Arxula adenine_orans LS3 have shown                      are carcinogenic compounds which are ubiquitous and
promise for BOD measurements even in salt water. This                    especially found around contaminated areas of closed-down
biosensor has been applied for BOD estimations of real                   gas works and cooking plants.
samples of coastal and island regions. Unlike the sensor
containing budding yeast which can also be used to                       Naphthalene being highly water soluble has been found in
measure BOD in salt water up to 10% (w/v); the mycelial                  contaminated soils. Amperometric biosensors for

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naphthalene were developed using either Sphingomonas sp.                in polyvinyl alcohol-bearing styrylpyridinium groups has
B1 or P. fluorescens WW4 cells immobilized within a                     shown promise. The measurements achieved with diuron
polyurethane-based hydrogel. These were tested in a flow-               and mercuric chloride has indicated future prospects of this
through system and a stirred cell (batch method) and were               bioassay for the detection of pollutants inhibiting
shown to be equally suited for the quantification of                    photosynthetic electron flow.
naphthalene in aqueous solutions. The sensors had an
operational lifetime of up to 20 days4. Other microbial                 Applications of microbial biosensors in food,
sensors were developed for xenobiotics such as phenol,                  fermentation and allied fields
chlorinated phenols, polychlorinated biphenyls, benzene, 3-             In recent years, the demand for quick and specific
chlorobenzoate. Acrylamide, acrylic acid and acrylonitrile              analytical tools for food and fermentation analysis has
are widely used in the chemical industry for the production             increased and is still expanding. Analysis is needed for
of various polymers, fibers and resins. Microbial assays,               monitoring nutritional parameters, food additives, food
which may have potential in the fabrication of biosensors,              contaminants, microbial counts, shelf life assessment and
have been reported for their quantification in the                      other characteristics like smell and odor. A variety of
wastewaters. Acrylonitrile, however, could be separately                sensors based on enzymes and antibodies as well as
measured using Pseudomonas pseudoalcaligenes2,3.                        electronic noses have been reported. Microbial biosensors
Organophosphorous compounds widely used as pesticides,                  have also shown potential in food analysis. Monitoring the
insecticides and chemical warfare agents have created                   quality of milk is an important parameter because present
public concern because of their widespread use and                      methods involve bulk collection and prolonged storage.
toxicity. A variety of enzyme sensors based on acetyl                   Rancidity or off-flavor in milk and milk products is caused
choline esterase5 and OPH7 have been reported. Biosensors               by the liberation of short-chain fatty acids (C4–C12).
based on genetically modified microbial cells with surface              Unlike other microorganisms, Arthrobacter nicotianae has
expressed OPH have been recently used in the construction               been shown to possess enzymes of the oxidation pathway
of potentiometric as well as a fiber-optic based microbial              with a high specificity towards short-chain fatty acids.
biosensor.                                                              These cells have been used in conjunction with an oxygen
                                                                        electrode for the fabrication of a microbial sensor in flow
Nitroaromatic compounds (nitrophenols, picric acid,                     injection analysis of short-chain free fatty acids in milk. A
trinitrotoluene and similar compounds) represent a wide-                number of reports are available on microbial biosensors for
spread group of xenobiotics present in wastes of chemical               amino acids such as tyrosine (Aeromonas phenologenes),
armament plants as well as civil factories manufacturing                tryptophan (P. fluorescens) and glutamic acid (B. subtilis).
dyes, pesticides and other chemicals. One of the major
microbial degradative products of nitroaromatics is nitrite             Microbial sensors have been developed for the
and this is used to monitor concentrations of such                      determination of vitamins such as vitamin B 12 (E.coli
pollutants. Nitrobacter sp. possessing high nitrite oxido-              L15) and ascorbic acid (Enterobacter agglomerans). Such
reductases has been described. Among them, the                          biosensor systems, for rapid determination of vitamin B 6,
mixotrophs have been characterized by high selectivity and              based on the respiratory activity of immobilized yeast
sensitivity to nitrite due to the induction of nitrite oxido-           (Saccharomyces u_arum) and Clark type oxygen electrode
reductase during mixotrophic cultivation. In contrast to                have been reported as rapid and simple determinations of
obligate autotrophs, the mixotrophic bacteria will not be               vitamin B 6 in marine products at ng/ml levels with good
liable to repression by organic compounds in the waste                  correlation to traditional microbial assay values.
sample. In this respect, the mixotrophic strain of N.ulgaris
has shown promise in the development of a biosensor                     Some of the environmental microbial biosensors discussed
possessing high selectivity with a lower limit of detection             above can also find applications in the determination of
of 10µ.                                                                 pesticides, insecticides, heavy metals and other chemical
                                                                        contaminants present in agricultural products or processed
The ability of cyanide to inhibit S. cerevisiae respiration             foods. Microbial sensors for the determination of sugars
has been utilized in developing a flow-type cyanide sensor.             such as glucose, sucrose and lactose and other compounds
The sensor was stable for about 16 days. A microbial                    like iron (T.ferrooxidans), ammonia (B.subtilis), acetic acid
sensor for measuring inhibitors and substrates for                      (Trichosporon brassicae), pyruvate, phosphate, peptide
nitrification in wastewater has been reported6. For the                 sweetener-aspartame, alcohol, etc. have also been reported.
determination of phytotoxicity, isolated chloroplasts or
photosynthetic membranes have been used. Major                          In pharmaceuticals and medicine, the microbial based
limitation of this approach, however, is the difficulty in              sensors have been studied for the estimation of steroids
their isolation. These problems have now been obviated by               such as cholesterol (Nocardia erythropolis) androstendione
the use of photosynthetic cyanobacterium such as                        (N. erythropolis), testosterone (N. erythropolis); antibiotics
Synechococcus sp.9,10. A microbial assay system based on                such as nystatin (S. cerevisiae) and other compounds like
photo-electrochemical cell for detecting pollutant-induced              gonadotropin releasing hormone (B. subtilis), urea
effects on the activity of the cyanobacterium immobilized

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(nitrifying bacteria), uric acid (Altenaria tennis), creatinine           respirometric analysis, although both techniques were
(Nitrobacter sp.) and iron (II and III) (T. ferrooxidans).                sensitive to the presence of pollutants. The toxicity of two
                                                                          common organotin pollutants and their initial breakdown
Applications of bioluminescence-based biosen-                             products (tributyltin, dibutyltin, triphenyltin and
sors                                                                      diphenyltin) were assessed in extracted soil solutions using
An important attribute of a biosensor is the real-time                    two different bioluminescent microbial biosensors:
process monitoring. This is necessary for monitoring                      Microtox and lux-modified P. fluorescens pUCD 6072. The
fermentors and wastewater biotreatment plants (WWBP).                     two biosensors showed different response patterns with
Prompt warnings of upsets in WWBP are important                           Microtox being more sensitive to the triorganotins and P.
because they circumvent the cost and time involved in                     fluorescens being more sensitive to the diorganotins.
reactivating or reinitiating the WWBP after shut down. A                  Bioluminescence has also been used for the detection of
frequent cause of upsets is the inflow of highly toxic                    GEM released in the environment. Solvents, crop
materials, including heavy metals and organic chemicals,                  protection chemicals etc. have also been monitored based
which exhibit toxicity to biota in a WWBP. The                            on E. coli heat shock promoters, fused with lux genes. A
bioluminescent reporter has advantageous properties such                  eukaryotic microbial biosensor for cytotoxicity analysis has
as rapid response, excellent sensitivity, large dynamic                   been recently reported. The yeast S. cerevisiae was
range and non-invasive continuous measurements                            genetically modified to express firefly luciferase,
amenable to automated data collection with minimal                        generating a bioluminescent yeast strain. This strain senses
manipulations.                                                            chemicals known to be toxic to eukaryotes in samples
                                                                          assessed as non-toxic by prokaryotic biosensors.
Accordingly, a microbial assay of metabolic death by loss
of bioluminescence from the marine microorganism                          In a typical study involving growth of E. coli using the lux
Photobacterium phosphoreum has been commercialized as                     phenotype as an indicator, it was observed that light
the Microtox system. Quantification using bioluminescent                  emission increased rapidly up to late log phase of growth.
microbial biosensors, which are based on heat shock gene-                 However, towards the end of cultivation, light emission of
bioluminescence gene fusions, has been proposed to be                     the cultures decreased to undetectable levels, but the colony
most suitable to face these challenges. Monitoring of the                 forming units were not affected. This indicated that despite
PAH-like naphthalene and salicylate using the genetically                 the cellular metabolic activity being completely absent,
modified P. fluorescens HK44 has been extensively                         cells retained their ability to be cultured17.
studied. This strain harbours the bioluminescent reporter
plasmid pUTK21 that contains a nahG-luxCDABE fusion                       Immobilization of biomaterial
in a salicylate inducible operon. The use of P. fluorescens               The basic requirement of a biosensor is that the biological
HK44 as a real time reporter has been demonstrated in both                material should bring the physico-chemical changes in
online and in situ studies. In October 1996, the US                       close proximity of a transducer. In this direction,
Environmental Protection Agency approved the release of                   immobilization technology has played a major role.
HK44 into contaminated soil ecosystems as a means of                      Immobilization not only helps in forming the required close
monitoring PAH bioavailability during a long-term                         proximity between the biomaterial and the transducer, but
bioremediation process. Procedures are being developed to                 also helps in stabilizing it for reuse. The biological material
enumerate HK44 cell numbers in soil under long-term                       has been immobilized directly on the transducer or in most
(over 2 years) field conditions.                                          cases, in membranes, which can subsequently be mounted
                                                                          on the transducer. Biomaterials can be immobilized either
Bioluminescent microbial biosensors have been extensively                 through adsorption, entrapment, covalent binding, cross-
investigated for the detection of heavy metals like Hg2+,                 linking or a combination of all these techniques. Selection
Zn2+, chromate–copper-arsenate, Ni2+ and chromate,                        of a technique and/or support would depend on the nature
antimonite and arsenate and Cd2+ and Pb2+. In recent years,               of the biomaterial and the substrate and configuration of the
the scientific validity for using total metal concentrations              transducer used.
that are measured using chemical methods, as a basis for
metal limits in soil, has been questioned. This is mainly due             The choice of support and technique for the preparation of
to the fact that a considerable amount of metal will be in a              membranes has often been dictated by the low diffusion
bound form rendering it non-available for biological                      resistance of the membrane. Gentle techniques need to be
processes. Hence luminescence-based microbial biosensors                  applied when viable cell preparations are to be used.
that are relevant to soil ecosystems have been developed                  Covalent binding, a commonly used technique for the
and have shown to be good predictors of the bioavailable                  immobilization of enzymes and antibodies, has not been
fraction of metals in soils.                                              useful for the immobilization of cells. One of the general
                                                                          problems with covalent binding is that the cells are exposed
The luminescent assay using genetically modified                          to potent reactive groups and other harsh reaction
(luminescence-marked) terrestrial bacteria was found to                   conditions thus affecting their viability. There may also be
have greater sensitivity and reproducibility than the                     a loss in the structural integrity of the cell during
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continuous use, leading to loss of intracellular enzymes.               Natural polymers used for the entrapment of the cells
Among others, is the very low cell loading, that is achieved            include alginate, carrageen, low-melting agarose, chitosan,
as compared to entrapment and other techniques.                         etc. These polymers are known to be very useful in
                                                                        obtaining viable cell-immobilized systems. Among these,
Cross-linking     using     bifunctional     reagents   like            entrapment in alginate by ionotropic gelation using a
glutaraldehyde has been successfully used for the                       variety of divalent and trivalent cations has found extensive
immobilization of cells in various supports. Of these,                  use in immobilized cell technology. The major limitations
proteinic supports such as gelatin, albumin and hen egg                 of Ca-alginate gels are their destabilization and subsequent
white have been extensively used. Even though this                      solubilization by the Ca-chelators present in the processing
technique obviates some of the limitations of covalent                  solution or waste. A novel technique has been developed
binding, the chemical cross-linking reagents used often                 for stabilizing the alginate membranes and beads towards
affect the cell viability. Thus cross-linking technique will            Ca chelators by reinforcing them with gamma ray
be useful in obtaining immobilized non-viable cell                      polymerized polyacrylamide. Major limitation of
preparations containing active intracellular enzymes. Stable            entrapment technique is the additional diffusion barrier
microbial preparations are often required for use under                 offered by the entrapment materials, which can be
varied environmental factors.                                           minimized by increasing the porosity of the matrix using
                                                                        open pore entrapment techniques9.
Cross-linking has been extensively used for the
stabilisation of enzymes. It has also been used for the                 Sensitivity of the enzyme sensors has been improved by
stabilization of cellular organelles to osmotic shock,                  combining various types of mediators with enzymes. In the
prevention of lysis of extremely halophilic cells in low salt           case of whole cells, the mediator should be able to shuttle
or salt free environments and the prevention of lysis of                the electrons between red-ox centers of intracellular
microbial cells by lytic enzymes present in the processing              enzymes and the electrode surface. Passive trapping of
streams14.                                                              cells into the pores or adhesion onto the surfaces of
                                                                        cellulose or other synthetic membranes has been well
Entrapment and adsorption techniques are more useful                    documented. The major advantage is that the cells
when viable cells are used. A common approach is to retain              immobilized through adhesion are in direct contact with the
the cells in close proximity to the transducer surface using            liquid phase containing the substrate, even though the cell
membranes like the dialysis membrane. In general, the                   and the liquid phase are distinctly separate thus reducing or
outer membrane must be chemically and mechanically                      eliminating the mass transfer problems commonly
stable, with a thickness of 10–15µ and a pore size of 0.1–              associated with gel entrapment methods.
1.0 µ. Microbial cells have been immobilized by
entrapment in a variety of synthetic or natural polymeric               A basic limitation of passive trapping or adhesion,
gels for use in industrial processing. Some of these                    however, is the possibility of cell wash out during
entrapment techniques have been used for biosensors.                    continuous use. The adhesion is found to be rapid and the
                                                                        cells adhere as a monolayer. The adhesion being very
The synthetic polymers used for microbial biosensor                     strong, the high ionic concentrations and extreme pH
applications include polyacrylamide, polyurethane-based                 conditions which normally disrupt the ionic interactions fail
hydrogels, photo cross-linkable resins and polyvinyl                    to desorb the cells. Cells can be adhered by coating the
alcohol. Polyvinyl alcohol is one of the most widely studied            cells, the supports or both with PEI. Viability of the cell
polymers, as it can form membranes, fibres, etc. Enzymes                were not affected by this treatment. A variety of cells
and cells have been immobilized in these membranes either               containing different enzymes including urease have been
by entrapment, covalent binding, cross-linking, freezing                immobilized using this technique and studies are underway
and thawing -irradiation, photo cross-linking or entrapment             for use of such bio-films in the fabrication of microbial
followed by cross-linking. However, the major limitation of             biosensors10.
synthetic polymer is the possible loss of viability of the
cell.                                                                   Biospecific reversible immobilization using lectins has
                                                                        been used for the introduction of biological catalysts into
This is more pronounced when polyacrylamide gels are                    analytical systems. The basic advantage is that when the
used. Photo cross-linkable polyvinyl alcohol bearing                    enzyme activity goes below the practical limits, the bound
styrylpyridinium groups has been shown to entrap cellular               enzyme can be easily eluted and the membranes or the
organelles and cells under very mild conditions, for use in             transducer surface can be loaded with a fresh batch of the
biosensors. Polyacrylonitrile membranes and albumin–poly                enzyme for reuse without significantly affecting the
(ethylene glycol) hydrogel, which have shown promise in                 transducer or membrane characteristics. Reversible
the immobilization of enzymes, may also gain importance                 immobilization through hydrophobic interaction has also
in microbial biosensors. Albumin–poly (ethylene glycol)                 shown promise. As microbial cell surfaces contain a
hydrogels, in view of their biocompatibility, may be useful             number of biospecific affinity binding or hydrophobic sites,
in the fabrication of in vivo implantable biosensors12.                 this approach can be used in the future for the reversible

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introduction of microbial cells on transducer surfaces.                   and low sensitivity and detection limits. Theoretical models
                                                                          for enzyme-based biosensors with nonreactive transducer
Future microbial biosensor design strategies                              predict that enzyme electrode sensitivity and detection limit
As the biosensor technology starts moving from the proof-                 can be improved by either lowering the KM or increasing
of-concept stage to field testing under realistic processes or            the bimolecular rate constant. The advancement in enzyme
waste monitoring conditions, the need for stable biological               engineering has made these goals possible through
materials will become important. Biomaterials that are                    approaches like site directed mutagenesis. Slow response of
stable and functional in highly acidic, alkaline,                         microbial sensors as compared to enzyme sensors has also
hydrophobic, saline, oxidizing, low and high temperature                  been attributed to diffusion problems associated with the
environments as well as immune to toxic substrates in the                 cell membranes. Possibility of genetically engineering the
processing stream will play an important role. A varied                   cell to express the enzymes of interest on cell surface can
population of microrganisms like the thermophiles,                        overcome this problem. Thus, it can be foreseen that
alkalophiles, halophiles, psychrophiles, metallophiles,                   microbial biosensors have been miniaturized extensively in
osmophiles, etc. will gain importance in the successful                   the recent years. Keeping in line with such developments,
applications of microbial sensors in realistic conditions.                microbial cells with high enzyme activities may be
Selective screening methods for discovering the rare genera               required. This is essential especially when microbial cells
and species of microbes containing novel analytically                     are used as substitutes to enzyme based sensors.
useful enzymes are essential. Equally important is the need               Recombinant DNA technology can help in the
for an integrated approach to combine classical                           overproduction of enzymes of interest in microbial cells or
microbiology with developments in modern biotechnology.                   in enhancing the specificities of microbial sensors by
Currently there is also an interest in cold active enzymes                activating certain pathways of metabolism and cellular
obtained from psychrophilic organisms exhibiting high                     uptake while switching off the undesirable ones.
activities even at low temperatures.
                                                                          There are interesting possibilities within the field of
Basic advantage of such enzymes/ organisms is the                         biosensors. Given the existing advances in biological
possibility of carrying out process monitoring even under                 sciences coupled with advances in various other scientific
chilled storage conditions normally practiced in food and                 and engineering disciplines, it is imminent that many
allied industries. Recently, a biosensor based on a                       analytical applications will be replaced by biosensors. A
psychrophilic strain of Deinococcus has been reported for                 fruitful fusion between biological sciences and other
the measurement of sugars. One of the major limitations in                disciplines will help to realize the full potential of this
the use of microbial biosensors, especially in waste                      technology in the future.
monitoring, is the heavy metal toxicity. Metal resistant
organisms would gain importance for such microbial                        References
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