Bioluminescence in

Environmental monitoring

Presented to :Ms Asifa Kiyani

Presented by: Nayyab Nadeem & Sara Hassan

AbstractBioluminescence is the production of light by living organisms such as bacteria,

fungi and animals .The following review deals with detection and monitoring of

pollutants using a biological method of rapid, cost effective and a sensitive

technique for toxicology testing is the latest idea. The past decade has developed

these novel tools which hold a far superior application compared to

conventional methods. Previously most such assays were plant, animal or algae

derived and as accurate as they were, they were expensive and required a large

sample size in order to be standardized. One of the firsts assays based on

bioluminescence was on the species V.fisheri and this has been reported to be

versatile and test protocol is simple.Bioluminescene based assays / techniques

are much sensitive when compared to wide range of chemicals or other bacterial

based assays such as enzyme inhibition, respirometry etc.The assays have the

added benefit of detecting toxicity across a wider range of toxic compounds.

Introduction• Bioluminescence in its simplest definition is the light emission by a living

organism.

• Their inherent beauty and eco friendliness has them marked for use in future

environmental monitoring ventures.

• However they can be time consuming and often rather daunting (3).Due to

these limitations the purpose is to find economic , quick and sturdy methods

of environmental monitoring and ecosystem maintainace.

• The toxicity assays utilizing these microorganisms are based on the working

principle that either light emission reduces or is inhibited in the presence of

pollutants they interfere with the metabolism of these microorganisms.

What is Bioluminescence

Naturally occurring bioluminescent

microorganisms in monitoring of

environment• The biolumiscenece emitting bacteria are found in freshwater,

deep seas and marine habitats. They belong to gram negative

cell wall group and are broadly divided into four types; vibrio,

shewanella, photobacterium and photorhabdus.

• The light emission results from metabolic reactions and the

process itself driven by co factors so when a particular toxic

substance interferes the light emission is compromised giving

an indication that a certain pollutant is present and extent to

which it is affected confirms the concentration of the pollutant.

• The bacterium photobacterium emits light

which lies in the blue green region of the

visible spectrum

• This method is sensitive, non invasive and

provides real time monitoring of pollutants.

V.fisheri

• The first isolated microorganism whose bioluminesing ability was

harnessed was vibrio fisheri

• The assay which was developed using a strain of V.fisheri was

later given the name Microtox . The system contains freeze-dried

bacteria which are activated prior to use and light emission is

measured by a luminometer.

• Hernando et al worked on v.fisheri strain and in 10 labs to

observe its light inhibition capabilities and assays based on

that. Their results were positive and confirmed that reliability

and the reproducibility of the assay designed based on light

inhibition of the V.fisheri.

• Scheerer et al carried out an experiment I n which they

provided optimum conditions for V.fisheri and cultures in

fermentors providing a long term continuous cultures for

reproducible detection of pollutants and their measurement

• The enzyme luciferase has two subunits which are alpha and beta

and coded by luxA and luxB

• This operons also consists of other gene such as luxC, lux D, luxE

and luxG along with luxA and luxB

• The greatest light emission occurs when cells are present in large

concentrations and not in dilute conditions

• Different mechanisms exist for the regulation of the expression of

the lux operons. The protein of luxR acts as a repressor.LuxI on the

other hand codes for autoinducer which leaves the cells and also

affects other surrounding cells. The repressor interacts with

autoinducer and results in decrease in light emission.

Bioluminescent organism used

in heavy metal monitoring and

detection• Heavy metal poisoning and its bioaccumulation is becoming an

arising concern.

• bioluminescent organisms may be used for the rapid remediation of

all such heavy metals polluted areas. These organisms usually have

promoters which are also called as sensing elements which allow

them to survive in such adverse environments.

• The luciferase genes are used as reported genes. The presence of

the analyte triggers a reaction which then results in light emission

• Angela et al constructed recombinant whole cell biosensors for the

detection and assessment of heavy metals. Both gram positive and

gram negative bacteria were used and were made to express the

bioluminescence producing genes luxCDABE in response to heavy

metals. Their results were supportive of the use of these microbial

recombinant whole cells.

• Sulivan et al worked with a primary statistical model based on the

crossings between the different detection ranges of five different

bioluminescent strains for identification of four heavy metals

cadmium, mercury, copper and arsenic. Their results showed that

the statistical model could be used with confidence and that no

sample pre treatment was required either.

• The use of these biosensors which use bioluminescent

microorganism as their biological agent have been used in

rainwater, freshwater and estuarine areas containing high

levels of mercury and they responded favorably.

• Another breakthrough which came was the online monitoring

of heavy metals in waste water effluents Waste water treated

effluents were found to contain heavy metals like lead ,

mercury etc and assays based on bioluminesing bacteria

coupled to devices have been developed which allow online

monitoring from remote far off areas of pollution.

Environmental monitoring by recombinant bioluminescent microorganisms

• One of the downside of the assays designed from V.fisheri is the

need for appropriate salt concentration due to their marine habitats

• They also function within a narrow pH and temperature range and

may not be suitable for terrestrial systems.

• E.coli is an example of transgenic bioluminescent microorganism

which has been used in evaluating pollutants in air, land and water

due to the vast genetic knowledge regarding these and they are

easy to use.

• One such example is a strain of E.coli called E.coli HB101 which

consists of the plasmid Pucd607 which consists of luxCDABE from

V.fisheri and the tet (tetracycline) promoter

• The other bacterial strains which have been transformed using this

Puc plasmid include pseudomonas fluroscens 10586 , P.putida F1

which is a bacterium able to breakdown toluene.

• The P.fluroscens is especially sensitive towards copper as well as

cadmium and has been used in monitoring of industrial effluents

such as malt whiskey distilleries.

• MacGrath et al reported that soils which had a low pH had high

levels of zinc and showed greater sensitivity towards the

p.fluroscens transgenic organism compared to E.coli HB101.

Why do we need Biomonitoring?

Water quality may be affected by:

Spills of oil and industrial products from tanks, pipelines

Pesticides from agricultural area, leaching pathogens

Endocrine disrupting chemicals

Neurotoxins, hepatotoxins from algae blooms

Contamination from terrorist attack (toxins, microbes,

viruses, radioactive compounds)

Accidents, sabotage etc.

MICROTOX ASSAY

Microtox assay uses marine bioluminescent bacterium strain Vibrio

fischeri (24)

V. Fischeri is exposed to a range of concentrations of toxic agents

like heavy metals Zn, Cd, Ni, Hg, Cu etc .

As the concentration of toxins in the sample increases the

consequential reduction in the light intensity emitted from the

bacterium is measured and compared to standard.

This shift in the light intensity output measured by a luminometer.

The concentration of the toxic substances fabricate a dose / light

response relationship.

Normal bioluminescence

Contaminated sample is added!!

Bioluminescence inhibition

Photobacterium Vibrio fischeri (Microtox test)

Photobacterium Vibrio fischeri

Bioluminescence

measurement at exposure

time 5, 15 and 30 minMicrotox M500 analyzer

Lights off lights on assay

Toxicity tests have been used for:

Marine and fresh waters biomonitoring

Toxicity testing of wastewaters and soils

Toxicity testing of fly ash leachates

Toxicity assessments of pure compounds, heavy metals and

pesticides

Wastewater treatment plant applications

Applications of ecotoxicity testing

Dinoflagellates and Bioluminescence

Dinoflagellates for environmental risk detection

• Bioluminescent dinoflagellates are also used in assays for ecotoxicological testing.

• The dinoflagellates Bioluminescence is dependant on circadian rhythms.

• In various studies Lingulodinium polyedrum, Pyrocystis noctiluca, Pyrocystis fusiformis, Ceratocorys horrida, Pyrophacus steinii and Pyrocystislunula have been employed to find the effects of xenobiotic contaminants on intensity of bioluminescence.

Use of dinoflagellates as a metal toxicity assessment tool in aquatic system

• Dinoflagellates selected Gonvaulax polvedra andPyrocystis lunula for the assessment of biologicaltoxicity of the selected contaminants present in theaquatic water samples. This study used thebioluminescence of dinoflagellates with QwikLiteassay developed by the US Navy for the heavy metalsHg, Cu, Cd, As, Pb, Cr, etc. The results obtained in theform of single metal toxicity in the order Hg+ > Cu > Cd> As > Pb > Cr Comparison was made to standardsolutions. Dinoflagellates demonstrated greatsensitivity to metal concentrations hence showinggreat prospect for testing heavy metal toxicity inaquatic systems (28).

ROLE OF BIOLUMINESENCE IN MARINE WATER AND WASTER

WATER TREATMENTS.

Assessment of heavy metals by bacterial bioluminescence in bench-scale wastewater treatment system

• According to one study the bioluminescent strain Shk1was used to determine the toxicity during the activatedsludge wastewater treatment in batch experiments usingbench-scale activated sludge system to determinepresence of heavy metals Ni, Cd, Cu and Zn using bothsources influent wastewater and activated sludge fromthe municipal wastewater treatment plant in batchexperiments.

• According to the findings of this study the Shk1 strainbioluminescence demonstrated the highest sensitivity toCd and Zn, then towards Cu, and least sensitivity to Ni.

USING OSTEOCOCCUS TAURI TO DETECT ANTIFOULING BIOCIDES IN MARINE WATER

• A recombinant was constructed using Ostreococcus tauri,known to be the smallest eukaryotic cell luminescentbiosensor with the aim to detect antifouling biocides.

• Diuron and Irgarol (antifouling biocides) and theirdegradation products prevalent in coastal waters wereselected to investigate the novel biosensor forecotoxicological testing.

• Cyclin-dependent kinase ( a cell cycle protein) fused toluciferase (CDKA-Luc) proved to be a highly sensitivebiosensors showing precise determination of diuron andIrgarol.

• Luminometer was employed to measure luminescencecompared to inhibition of growth. Luminescence is a moresensitive indicator of toxicity than growth inhibition in marinephytoplankton.

Detection of drugs in surface water and wastewater samples preliminary testing of

toxicity studies using vibrio fischeri

• Effluent from various industries including municipal waste waterfind their final resting place in the large water bodies like seas andoceans.

• To detect these pharmaceutical compounds in the water sample apreliminary test is performed using the bioluminescent strain VibrioFischeri for executing Microtox®.

• detect the presence of analgesic drugs, primarily acidic and polar innature. The drugs investigated according to a particular study wereibuprofen, gemfibrozil, diclofenac, naproxen, with theirdecomposition products like salicylic acid.

• This preliminary step was followed by combined analytical methodusing (LC–ESI-MS) toxicity and liquid chromatography–electrosprayionization-mass spectrometry for determination of pharmaceuticalcompounds in the water samples

GENETICALLY ENGINEERED MICROORGANISMS FOR

POLLUTION MONITORING

Immobilization and integration into biosensors

No matter the type of modification used the bacteriareporter strain has to be sophisticated and for itsspecific use it must be immobilized to a bioactivemembrane to achieve and attain its final purpose ofdetection of whatever particular type of substance ithas been tailored for .So for this purpose it is take awayfrom its boundaries of the lab and taken to the beadhered or incorporated into a biosensor divide thatensures protection and maintenance along with easeof storage ease of sample access and transduction ofsignals. For this it may be impregnated into strontiumalginate. onto optic fiber tips (48) immobilization inagar (49)and encapsulation in gel matrices

bioavailability of nutrients.

• After observing the phenomenon of eutrophication ofalgal blooms which occur in the presence of nitrogen andphosphorus in excess a novel branch of bioreporters wasconstructed which worked on the principle of nutrientbioavailability. Nitrogen and phosphorus clearly don’tmake it t the top of the most deleterious pollutants listbut the principle of eutrophication was used to tailor

• Synechococcus sp. with a glnA::lux fusion and aSynechocystis sp containing a nblA::lux fusion each servesas a sensitive reporters nitrogen bioavailability. Theformer is specific for ammonia, nitrite and organicnitrogen on the contrary the latter is specific for nitratealone.

Detection of specific pollutants

• The first constructs to detect specific inorganic and organic pollutants fordetermining the bioavailability and catabolic activity and potential inwater bodies like streams and rivers a genetically modified bioluminescentbacteria Psuedomonas flouresens HK44 was employed.

• It bears a fused transcriptional fragment nahG-"uxCDABE responsible fornaphthalene and salicyclate catabolism.

• exposure of either compounds induced bioluminescence. The cells of P.flouresens were immobilized in strontium alginate to make an opticalbased whole-cell biosensor for monitoring of bioavailability ofnaphthalene and salicylate in streams of waste water.

• On exposure to additional compounds for instance glucose,toluene,complex nutrient medium either showed nill or weak bioluminescenceincreases produced after delayed response times compared tonaphthalene.

References

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