heavy metal detection using bioluminescence

7/30/2019 heavy metal detection using bioluminescence

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Under the Guidance of:

Swetha BN, Lecturer, RISMStudents Working:-

Kaustubh Parekh,

Ashwini Takalkar,

Moumita Bhattacharyya,

Amrutha Valli.


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IntroductionBioluminescence is the production and

emission of light by a living organism.

Its name is a hybrid word, originating

from the Greek bios for "living" and theLatin lumen "light".

Bioluminescence is a naturally

occurring form of chemiluminescence

where energy is released by a chemicalreaction in the form of light emission.

Fireflies, anglerfish, and other creatures

produce the chemicals luciferin (a

pigment) and luciferase (an enzyme).


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Biochemistry of Bioluminescence


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Bioluminescence has several functions in different taxa:

Counterillumination camouflage

Mimicry to attract prey

Attracting mates

Distraction

Repulsion of Predators

Communication

Illumination


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Bioluminescence in BiotechnologyBioluminescent organisms are a target for many areas of

research.

Luciferase systems are widely used in the field of genetic

engineering as reporter genes.Luciferase systems have also been harnessed for biomedical

research using bioluminescence imaging.

Bioluminescence is used to determine the amount of toxic

chemicals present in water.


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Bioluminescence & Water pollution-

The Overview

One would be able to determine the extent of

pollution by measuring how quickly the light dims asthe chemicals kill samples of Bioluminescent bacteria.


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Bioindicators Bioindicators are microorganism which acts as indicatorsof several substances usually presence of pollutant inwater, land, air etc.

These microorganisms can be used to make biosensors.A biosensor is a device that detects, records, and transmits

information regarding a physiological change or thepresence of various chemical or biological materials in theenvironment.

More technically, a biosensor is a probe that integrates abiological component, such as a whole bacterium or abiological product (e.g., an enzyme or antibody) with anelectronic component to yield a measurable signal.


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Photobacterium phosphoreum

Photobacterium phosphoreum or Vibrio phosphoreum is aGram-negative bioluminescent bacterium living in symbiosis

with marine organisms. It can emit bluish-green light (490 nm)due to a chemical reaction between luciferin and molecularoxygen catalysed by an enzyme called Luciferase.


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Photobacterium phosphoreum has been used because of its reliable

bioluminescence to measure toxicity in aquatic environments due to

biodegradable water toxins. The responsibility, simple operation, and costperformances of bioluminescence makes it a reliable reporter for assessing

aquatic samples containing toxicants such as pesticides, PCBs, aromatic

hydrocarbons, fuels, alkanes, alcohols, and heavy metals.


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Hypothesis Drinking water in and around Bangalore is highly

contaminated.

Severe effect include arthritis, diseases of kidney,circulatory system, nervous system etc.

Traditional approach for water quality testing involvesstandard analytical procedures

Most of them are laborious and time consumingAlternative to specific chemical method is bioassay


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Aim:-

Detecting heavy metals by Photobacteriumphosphoreum


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Objectives Isolation and culturing ofphotobacterium phosphoreum

Biochemical Characterization ofphotobacterium

phosphoreum

Comparison of bioluminescence of different water samples

Analysis of luminescence values.

Assessing the toxicity of water using isolated organism.


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METHODOLOGYThe Bangada fish was brought from the local market of

dwarakanagar, and use as the source

Isolation ofBioluminescent organism Enrichment

Isolation

Subculture


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Colony characteristics Shape -Circular

Margin -Entire

Elevation -Raised

Pigment -No pigment

Staining techniques

Gram staining -Gram negative

Negative staining -No capsule

Endospore staining- -No endospore

Capsule staining -No capsule


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Biochemical characterization

Test Results

Indole test -Negative Catalase test -positive

Oxidase test -Negative

Sucrose utilization test -Negative


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Gram -ve Indole test

Catalase test

BIOCHEMICAL TESTS RESULTS


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Comparison of bioluminescence of different watersamples.


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Sequencing Result>KAMA

GGCGGAGGGATCGGATTACTGGGCGTAAGCGCATGCAGGCGGTCTGTTAAGCAAGATGTGAAAGCCCGGGGCTCAACCTCGGAACAGCATTTTGAACTGGCAGACTAGAGTCTTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGAAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACGCGTTAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAATTCGCTAGAGATAGCTTAGTGCCTTCGGGAA

CTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCACGTAATGGTGGGAACTCCAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGTATACAGAGGGCTGCAAGCTAGCGATAGTGAGCGAATCCCACAAAGTACGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTTACCACGGTGTGGTTCATGACTGGGGTGAGCGAG


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Luminescence Spectrometric

readings at 490 nm


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0

100

200

300

400

500

600

0 2000 4000 6000 8000 10000 12000

Spectrometer

Reading

Concentration of Heavy metals (ppm)

mercury cobalt insecticide

x

y


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0 0.01 0.010.06

0.26

0.44

0.55

0.620.68

0.74

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

1 2 3 4 5 6 7 8 9 10

ABSORBANCEAT700NM

TIME IN HOURS

BACTERIAL TOLERANCE TEST


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Summary Effects of Heavy metals on production of

bioluminescence was studied

This method is comparable with these old methodsbecause they are complex, time consuming, expensiveand require sample pretreatment

Novelty of project


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REFERENCES 1.Broers, c.a.m1 and Lappalainen j, New developments in the

bioluminescence assay, Courrier du Savoir, pp. 107-110, 2004 2.Kenneth Wm. Thomulka and Lois H. Peck, Use of Bioluminescence

in Detecting Biohazardous Substances in Water, Philadelphia Collegeof Pharmacy and Science, pp.219-227

3.Attar H, Afshar S., Design of sensible biosensor for rapid detection ofbiocides in poble water, Asian journal of Biotechnology(2), pp. 120-126,2010

4.Almog R.,Daniel R., Melamed S.,Belkin S., bioluminescentwhole - cell biosensor for on-line water toxicity detection, TwelfthInternational Conference on Miniaturized Systems for Chemistry and

Life Sciences, pp.597-599, 2008 5.Lojek A., Ciz M., Kubala L., The use of terrestrial bacteria with

natural bioluminescence in environmental toxicology, Chem. Listy 101,pp.s116-s118,2007

6. Christine J. K., Nattapong T., Curtis A. L., Assessing wastewatermetal toxicity with bacterial bioluminescence in a bench-scale

wastewater treatment system, 2003


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7.Lapota, D., Moskowitz, G. J., Rosenberger, D. E., and Grovhoug, J. G., The Use of

Stimulable Bioluminescence from Marine Dinoflagellates as a Means of Detecting Toxicityin the Marine Environment,, Environmental Toxicology and Risk Assessment: 2nd Volume,pp. 1-158 . Chen A. Letz M, The Use of Dinoflagellate Bioluminescence to Characterize Cell

Stimulation in Bioreactors biotechnology and bioengineering, VOL. 83, NO. 1, pp. 93-103,2003

9. Karsi A.,Howe K.,, Wills R., Bailey R.H., Development of bioluminescent Salmonellastrains for use in food safety, BMC Microbiol. 200810.Lloyd D., James C.J., Hastngst J.W., Oxygen Affinities of the Bioluminescence Systems of

Various Species of Luminous Bacteria, Journal of General Microbiology, 131, 2137-2140, 198311. Water P., Lloyd D., Salt, pH and Temperature Dependencies of Growth andBioluminescence of Three Species of Luminous Bacteria Analysed on Gradient Plates,Journal of General Microbiology, 131, pp.2865-2869, 198512. Yagi K., Applications of whole-cell bacterial sensors in biotechnology andenvironmental science, Appl Microbiol Biotechnol, 2006.13. Eltzov E., Prilutsky E., Kushmaro A., Marks R.S., Geddes C.D., Metal-enhancedbioluminescence: An approach for monitoring biological luminescent processes, applied

physics letters pp.94. 083901-1-3, 2009 .

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heavy metal detection using bioluminescence