Microorganisms as Bio Indicators and Biosensors

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Microorganisms as bio indicators

and biosensors

Parvaiz ahmad ganie

AEM MA2 01


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What are bioindicators

Bioindicators are organisms, chemical markers or biological processeswhose change point can be observed to altered environmental conditionsand can be used to identify and quantify the effects of pollutants on theenvironment.

It can also be defined as anthropogenically induced response inbiomolecular, biochemical and physiological effects on one or moreorganisms, population, community or ecosystem level of biologicalorganization.

Bioindicators can tell us about the cumulative effects of differentpollutants in the ecosystem and about how long a problem may persist

for example:-

a) abundance of large marine organism or darkening of coralpigmentation may indicate that a reef has been exposed to poor quality ofwater for several weeks or months

b) reduced photosynthesis in plants or coral may indicate stress due toexposure of herbicides.


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Design of a bio indicator


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Criteria for selecting bioindicators :

Indicator should have casual relationship to ecological significantendpoint.

Indicator should have specific dose responsiveness to specificstressor i.e. should be sensitive and specific

Indicator should have wide temporal and spatial distribution.

Indicator should be available all the year and should have lowvariability to noise in the system.

Indicator should have results which are transparent andreproducible.

Indicator should sometime even surrogate the role of other

responses. Indicator should be easy to collect and should be cost-effective


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Types of bio indicators


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Cont .

Bioindicators may be of two types

accumulation bioindicator: store pollutants without any evidentchanges in their metabolisms;

response bioindicator: react with cell changes or visible symptomsof damage when taking up even small quantity of harmful

substances. Types of responses observed while using them may be:

ecological changes: involving changes in population density,

key species and species diversity;

behavioural changes: can be changes in feeding activities, bacterial

mobility or web spinning;

physiological changes: can be accumulation of heavy metal, CO2

production, BOD and microbial activity.


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Microorganisms as bioindicators :

Microorganisms are diverse group of organisms

found in large quantities and are easier to detect

and sample.

The presence of some microorganisms is well

correlated with particular type of pollution and it

serves as standard indicator of pollution.

Some bacteria produce stress proteins inresponse to contaminant like cadmium and

benzene.


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Bacterial characteristics for water analysis

The indicator bacterium should be present wheneverenteric pathogens are present.

The indicator bacterium should not reproduce incontaminated water and produce inflated values.

It should survive longer than the hardiest entericpathogen.

It should have greater specificity.

Its detection assay should be easy to perform.

It should be harmless to humans. Its level in water should have some direct relationship

with faecal pollution.


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Some bio indicators indicating status of

aquatic systems

Micro organism/bacteria Status of aquatic system

Escherichia coli Faecal origin

Streptococcus faecalis -do-

kliebsella -do-

Clostridium perfringens -do-

C.perfringens -do-

Spirillium volutants

pores

Industrial chemicals and toxic chemical wastes

Thiothrix Effulent from oil refineries contaning dehydrapen

sulphide


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Cont.

Micro organism/bacteria Status of aquatic system

Vogesella indigofera heavy metal contamination

Vibrio harveyi Mutagenic pollution


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Bioluminescent bacteria as bio

indicators

Bioluminescent bacteria: These are used to testwater for environmental toxins

If there are toxins present in the water, thecellular metabolism of bacteria is inhibited ordisrupted.

This affects quality or amount of light emitted bybacteria

It is very quick method and takes just 30 minutesto complete but could not identify the toxin

Examples of such luminous bacteria includePhotobacterium fisceri, P. phosphoreum


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Assay to check for the presence of toxic contaminant using

bioluminescent bacteria


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Microbes as biosensors


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Introduction

It is an analytical device which converts abiological response into an electrical signal.

It detects, records, and transmits informationregarding a physiological change

or process.

It determines the presence and concentration ofa specific substance in any test solution.


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Basic components

Bio-element

Transducer component


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Bioelement

It is a typically complex chemical systemusually extracted or derived directly from abiological organism.

Types :

Enzymes AntibodiesOxidase TissuePolysaccharide Nucleic Acid


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Bio-elemet

Function

To interact specifically with a target

compound i.e. the compound to be detected.

It must be capable of detecting the presence of

a target compound in the test solution.

The ability of a bio-element to interactspecifically with target compound (specificity)

is the basis for biosensor.


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Transducer

Function :

To convert biological response in to anelectrical signal.

Types :

Electrochemical,

Optical,

Piezoelectric


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Working of bio sensor

Figure. Schematic Diagram of Biosensor

a- Bio-elementb- Transducerc- Amplifierd- Processore- Display


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Response of a bio element

Heat absorbed (or liberated ) during the

interaction.

Movement of electrons produced in a radox

reaction.

Light absorbed (or liberated ) during the

interaction.

Effect due to mass of reactants or products


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Types of bio sensors

Electrochemical biosensor

Optical biosensor

Thermal biosensor

Resonant biosensor

Ion-sensitive biosensor


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MICROBIAL BIOSENSORS

A biosensor is a device that detects, transmits and recordsinformation regarding a physiological or biochemical change.

Technically, it is a probe that integrates a biological component withan electronic transducer thereby converting a biochemical signal

into a quantifiable electrical response. Biosensors make use of a variety of transducers such as

electrochemical, optical, acoustic and electronic

The function of a biosensor depends on the biochemical specificityof the biologically active material.

The choice of the biological material will depend on a number offactors viz the specificity, storage, operational and environmentalstability.


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Selection also depends on the analyte to be detectedsuch as chemical compounds antigens, microbes,hormones, nucleic acids or any subjective parameterslike smell and taste.

Enzymes, antibodies, DNA, receptors, organelles andmicroorganisms as well as animal and plant cells ortissues have been used as biological sensing elements.

Some of the major attributes of a good biosensingsystem are its specificity, sensitivity, reliability,

portability, (in most cases) ability to function even inoptically opaque solutions, real-time analysis andsimplicity of operation.


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Use of microbial cells as biosensing

elements Advantages of microbes as biological sensing materials in

the fabrication of biosensors.

Present ubiquitously

Able to metabolise a wide range of chemical compounds.

Great capacity to adapt to adverse conditions Develop the ability to degrade new molecules with time

Microbes are also amenable for genetic modificationsthrough mutation or through recombinant DNA technology

Serve as an economical source of intracellular enzymes


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CONT

In the construction of biosensors purifiedenzymes have been most commonly used due to

their high specific activities as well as

high analytical specificity. Limitation

expensive

unstable, Over 90% of the enzymes known to date are

intracellular


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In this respect, the utilisation of whole cells as a source ofintracellular enzymes has been shown to be a better alternative topurified enzymes in various industrial processes (Bickerstaff, 1997;DSouza, 1999).

why so???

Because It avoids the lengthy and expensive operations of enzyme

purification

preserves the enzyme in its natural environment

protects it from inactivation by external toxicants such as heavy

metals Whole cells also provide a multipurpose catalyst especially when

the process requires the participation of a number of enzymes insequence


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Whole cells ~ usage ~viable or non-viable form

Viable cells are gaining considerable importance in the fabrication ofbiosensors (Burlage and Kuo, 1994; Riedel, 1998; Arikawa et al.,1998;Simonian et al., 1998).

Why?

Viable microbes metabolise various organic compounds eitheranaerobically or aerobically resulting in various end products likeammonia, carbon dioxide, acids etc that can be monitored using a varietyof transducers.

Viable cells are mainly used when the overall substrate assimilationcapacity of microorganisms is taken as an index of respiratory metabolicactivity, as in the case of estimation of biological oxygen demand (BOD) or

utilisation of other growth or metabolically related nutrients likevitamins,sugars, organic acids and nitrogenous compounds(Riedel, 1998).Another mechanism used for the viable microbial biosensor involves theinhibition of microbial respiration by the analyte of interest, likeenvironmental pollutants (Arikawa et al., 1998).


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Limitation

Diffusion of substrate and products through the cell wall resulting ina slow response as compared to enzyme- based sensors (Rainina etal., 1996).

One of the ways to obviate this problem is to use permeabilised

cells. Permeablisation can be achieved via

physical (freezing and thawing),

chemical (organic solvents/detergents) and

enzymatic (lysozyme, papain) approaches

The most common technique uses organic solvents such as toluene,chloroform, ethanol and butanol or detergents like N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB), Na-deoxycholate anddigitonin (Patil and DSouza, 1997).


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Such chemical treatment creates minute pores byremoving some of the lipids from the cellmembranes, thereby allowing for the freediffusion of small molecular weight substrates/products across the cell membrane whileretaining most of the macromolecularcompounds like the enzymes inside the cell.

The permeabilisation process, however, rendersthe cell non-viable but can serve as aneconomical source of intracellular enzymes.


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In the case of periplasmic enzymes such as invertase and catalase in yeast (DSouzaand Nadkarni, 1980; Svitel et al., 1998) and urease and phosphatases in bacteria(Kamath and DSouza, 1992; Macaskie et al.,1992) whole cells can be used withoutpermeabilisation

One of the recent advances is to engineer the cell to transport the intracellularenzyme and anchor it into the periplasmic space.

Such an approach has been applied to obtain recombinant Escherichia colicellswith surface expressed oragnophosphorous hydrolase(OPH), an enzyme useful inthe fabrication of biosensors for the detection of organophosphate compounds(Mulchandani et al., 1998a,b).

These cells could degrade the organophosphates more efficiently (Mulchandani etal., 1998a,b) without the diffusional limitations otherwise observed in engineeredcells expressing OPH intracellularly (Rainina et al., 1996).

The above approach is an important development in the field of microbialbiosensors as it provides a cell system with no membrane transport problems andat the same time will not affect the cellular structure and activity.

This is in contrast to chemically permeabilised cells which result in loss of cellviability


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These types of genetic approaches may have

major significance in the future, especially for

sensors like BOD wherein polymers such as

protein, starch, lipid etc have to be brokendown to monomers before they can be

metabolised.


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Another limitation in using whole cells is the low specificity ascompared to biosensors containing pure enzymes.

This is mainly due to the unwanted side reactions catalysed byother enzymes in a cell.

Several approaches are being investigated to minimise such non-

specific reactions. Permeabilisation of the cell empties it of most of the small

molecular weight cofactors etc, thus minimizing the unwanted sidereactions (DSouza, 1989a).

Thus a whole cell of yeast containing intracellular -galactosidaseconverts lactose to ethanol and CO2 whereas the same cell onpermeabilisation converts lactose only to glucose and galactose dueto the loss of cofactors from the cell (Rao et al., 1988; Joshi et al.,1989).


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Side reactions, which can occur due to the presence ofother enzymes in a cell, can also be minimised byinactivating such enzymes either by physical (heat) orchemical means when non-viable cells are used (Godboleet al., 1983; Di Paolantonio and Rechnitz, 1983; DSouza,

1989a; Riedel, 1998). Another approach that is of significance in viable cell-

based biosensors is the blockage of unwanted metabolicpathways or transport systems.

Thus, for the determination of glutamic acid in thepresence of glucose by Bacillus subtilis, the glucose uptakecarrier system of the cell was blocked using a thiol inhibitorlike chloromercuribenzoate and also the glycolysis wasreversibly inhibited by NaF (Riedel and Scheller, 1987). .


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Microbial biosensors based on light emission fromluminescent bacteria are being applied as a sensitive,rapid and non-invasive assay in several biologicalsystems (Burlage and Kuo, 1994; Matrubutham andSayler, 1998).

Bioluminescent bacteria are found in nature, theirhabitat ranging from marine (Vibrio fischeri) toterrestrial (Photorhabdus luminescens) environments.

Bioluminescent whole cell biosensors have also been

developed using genetically engineeredmicroorganisms (GEM) for the monitoring of organic,pesticide and heavy metal contamination.


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The microorganisms used in these biosensors aretypically produced with a constructed plasmid inwhich genes that code for luciferase are placedunder the control of a promoter that recognisesthe analyte of interest.

When such microbes metabolise the organicpollutants, the genetic control mechanism also

turns on the synthesis of luciferase, whichproduces light that can be detected byluminometers.


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One approach to environmental monitoring is to detectchanges in gene expression patterns induced byadverse conditions.

Bacterial strains that increase light production in the

presence of specific chemicals have been constructedusing bioluminescence genes (lux) as reporters oftranscriptional responses.

A typical example is the Pseudomonas fluorescensHK44, a lux-based bioluminescent bioreporter that is

capable of emitting light upon exposure tonaphthalene, salicylate and other substitutedanalogues.


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Immobilisation of bio materials

The basic requirement of a biosensor is that the biological materialshould bring the physico-chemical changes in close proximity of atransducer.

Immobilisation not only helps in forming the required closeproximity between the biomaterial and the transducer, but also

helps in stabilising it for reuse. The biological material has been immobilised directly on the

transducer or in most cases, in membranes, which cansubsequently be mounted onthe transducer.

Biomaterials can be immobilised either through adsorption,entrapment, covalent binding, cross-linking or a combination of all

these techniques(DSouza, 1989a, 1999; Bickerstaff, 1997).

e.g; Covalent binding, commonly used technique for theimmobilisation of enzymes and antibodies.


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Microbial biosensors for

environmental applications


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Cont..


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Applications of bioluminescence-

based biosensors


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Refrences

Bickerstaff, G.F. (Ed.), 1997. Immobilization of Enzymes and Cells.Humanae Press, Totowa, NJ. Burlage, R., Kuo, C.T., 1994. Living biosensors for the management and manipulation of microbial

consortia. Annu. Rev. Microbiol. 48, 291309

DSouza, S.F., 1989a. Immobilized cells: techniques and applications. Indian J. Microbiol. 29, 83117.

Godbole, S.S., DSouza, S.F., Nadkarni, G.B., 1983. Regeneration of NAD(H) by alcoholdehydrogenase in gel-entrapped yeast cells. Enzyme Microb. Technol. 5, 125128.

Joshi, M.S., Gowda, L.R., Katwa, L.C., Bhat, S.G., 1989. Permeabilization of yeast cells (Kluyeromycesfragilis) to lactose by digitonin. Enzyme Microb. Technol. 11, 439443

Matrubutham, U., Sayler, G.S., 1998. Microbial biosensors based on optical detection. In:Mulchandani, A., Rogers, K.R. (Eds.), Enzyme and Microbial Biosensors: Techniques andProtocols.Humanae press, Totowa, NJ, pp. 249256

Riedel, K., 1998. Microbial biosensors based on oxygen electrodes. In: Mulchandani, A., Rogers, K.R.(Eds.), Enzyme and Microbial Biosensors: Techniques and Protocols. Humanae Press, Totowa, NJ,pp. 199223.

Svitel, J., Curilla, O., Tkac, J., 1998. Microbial cell-based biosensor for sensing glucose, sucrose orlactose. Biotechnol. Appl. Biochem. 27, 153158.


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Microorganisms as Bio Indicators and Biosensors

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