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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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