Bioluminescence, the production of light by living organisms, can be used to sensitively monitor pollution through assays that detect changes in bioluminescence caused by toxic substances. One of the earliest assays was developed using the bacterium Vibrio fisheri, measuring inhibition of its bioluminescence by pollutants. Recombinant bacteria like E. coli transformed with bioluminescence genes have also been used for biomonitoring due to their genetic tractability and ability to detect a wide range of pollutants in various environments. Dinoflagellates exhibiting bioluminescence have further been employed for toxicity assessments of metals and other contaminants in aquatic systems.

1. Bioluminescence in
Environmental monitoring
Presented to :Ms Asifa Kiyani
Presented by: Nayyab Nadeem & Sara Hassan

2. Abstract
Bioluminescence 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.

3. 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.

4. What is Bioluminescence

5. 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.

6. • 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.

8. 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.

9. • 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

10. • 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.

11. 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

12. • 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.

13. • 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.

14. 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.

15. • 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.

16. 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.

17. 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.

18. Photobacterium Vibrio fischeri (Microtox test)
Bioluminescence inhibition
Normal bioluminescence
Contaminated sample is added!!
Photobacterium Vibrio fischeri
Bioluminescence
measurement at exposure
Microtox M500 analyzer time 5, 15 and 30 min

20. Lights off lights on assay

21. Applications of ecotoxicity testing
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

22. Dinoflagellates and Bioluminescence

23. 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 Pyrocystis
lunula have been employed to find the effects of
xenobiotic contaminants on intensity of
bioluminescence.

25. Use of dinoflagellates as a metal toxicity
assessment tool in aquatic system
• Dinoflagellates selected Gonvaulax polvedra and
Pyrocystis lunula for the assessment of biological
toxicity of the selected contaminants present in the
aquatic water samples. This study used the
bioluminescence of dinoflagellates with QwikLite
assay developed by the US Navy for the heavy metals
Hg, Cu, Cd, As, Pb, Cr, etc. The results obtained in the
form of single metal toxicity in the order Hg+ > Cu > Cd
> As > Pb > Cr Comparison was made to standard
solutions. Dinoflagellates demonstrated great
sensitivity to metal concentrations hence showing
great prospect for testing heavy metal toxicity in
aquatic systems (28).

26. ROLE OF BIOLUMINESENCE IN
MARINE WATER AND WASTER
WATER TREATMENTS.

27. Assessment of heavy metals by
bacterial bioluminescence in bench-scale
wastewater treatment system
• According to one study the bioluminescent strain Shk1
was used to determine the toxicity during the activated
sludge wastewater treatment in batch experiments using
bench-scale activated sludge system to determine
presence of heavy metals Ni, Cd, Cu and Zn using both
sources influent wastewater and activated sludge from
the municipal wastewater treatment plant in batch
experiments.
• According to the findings of this study the Shk1 strain
bioluminescence demonstrated the highest sensitivity to
Cd and Zn, then towards Cu, and least sensitivity to Ni.

28. USING OSTEOCOCCUS TAURI TO DETECT ANTIFOULING
BIOCIDES IN MARINE WATER
• A recombinant was constructed using Ostreococcus tauri,
known to be the smallest eukaryotic cell luminescent
biosensor with the aim to detect antifouling biocides.
• Diuron and Irgarol (antifouling biocides) and their
degradation products prevalent in coastal waters were
selected to investigate the novel biosensor for
ecotoxicological testing.
• Cyclin-dependent kinase ( a cell cycle protein) fused to
luciferase (CDKA-Luc) proved to be a highly sensitive
biosensors showing precise determination of diuron and
Irgarol.
• Luminometer was employed to measure luminescence
compared to inhibition of growth. Luminescence is a more
sensitive indicator of toxicity than growth inhibition in marine
phytoplankton.

29. Detection of drugs in surface water and
wastewater samples preliminary testing of
toxicity studies using vibrio fischeri
• Effluent from various industries including municipal waste water
find their final resting place in the large water bodies like seas and
oceans.
• To detect these pharmaceutical compounds in the water sample a
preliminary test is performed using the bioluminescent strain Vibrio
Fischeri for executing Microtox®.
• detect the presence of analgesic drugs, primarily acidic and polar in
nature. The drugs investigated according to a particular study were
ibuprofen, gemfibrozil, diclofenac, naproxen, with their
decomposition products like salicylic acid.
• This preliminary step was followed by combined analytical method
using (LC–ESI-MS) toxicity and liquid chromatography–electrospray
ionization-mass spectrometry for determination of pharmaceutical
compounds in the water samples

30. GENETICALLY ENGINEERED
MICROORGANISMS FOR
POLLUTION MONITORING

31. Immobilization and integration into
biosensors
No matter the type of modification used the bacteria
reporter strain has to be sophisticated and for its
specific use it must be immobilized to a bioactive
membrane to achieve and attain its final purpose of
detection of whatever particular type of substance it
has been tailored for .So for this purpose it is take away
from its boundaries of the lab and taken to the be
adhered or incorporated into a biosensor divide that
ensures protection and maintenance along with ease
of storage ease of sample access and transduction of
signals. For this it may be impregnated into strontium
alginate. onto optic fiber tips (48) immobilization in
agar (49)and encapsulation in gel matrices

32. bioavailability of nutrients.
• After observing the phenomenon of eutrophication of
algal blooms which occur in the presence of nitrogen and
phosphorus in excess a novel branch of bioreporters was
constructed which worked on the principle of nutrient
bioavailability. Nitrogen and phosphorus clearly don’t
make it t the top of the most deleterious pollutants list
but the principle of eutrophication was used to tailor
• Synechococcus sp. with a glnA::lux fusion and a
Synechocystis sp containing a nblA::lux fusion each serves
as a sensitive reporters nitrogen bioavailability. The
former is specific for ammonia, nitrite and organic
nitrogen on the contrary the latter is specific for nitrate
alone.

33. Detection of specific pollutants
• The first constructs to detect specific inorganic and organic pollutants for
determining the bioavailability and catabolic activity and potential in
water bodies like streams and rivers a genetically modified bioluminescent
bacteria Psuedomonas flouresens HK44 was employed.
• It bears a fused transcriptional fragment nahG-"uxCDABE responsible for
naphthalene and salicyclate catabolism.
• exposure of either compounds induced bioluminescence. The cells of P.
flouresens were immobilized in strontium alginate to make an optical
based whole-cell biosensor for monitoring of bioavailability of
naphthalene and salicylate in streams of waste water.
• On exposure to additional compounds for instance glucose,toluene,
complex nutrient medium either showed nill or weak bioluminescence
increases produced after delayed response times compared to
naphthalene.

34. References
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T., Belkin, S., 2003, Fluorescence and bioluminescence reporter functions in
genetically modified bacterial sensor strains, Sens. Actuators B-Chem. 90:2–8.
• Sayler, G. S., Simpson, M. L., Cox, C. D., 2004, Emerging foundations: nano-engineering
• and bio-microelectronics for environmental biotechnology, Curr. Opin. Microbiol.
7:267– 273.
• Selifonova, O., Burlage, R., Barkay, T., 1993, Bioluminescent sensors for detection
of bioavailable Hg(II) in the environment, Appl. Environ. Microbiol. 59:3083–3090.
• Shao, C. Y., Howe, C. J., Porter, A. J. R., Glover, L. A., 2002, Novel cyanobacterial
biosensor for detection of herbicides, Appl. Environ. Microbiol. 68:5026–5033.
• Stiner, L., Halverson, L. J., 2002, Development and characterization of a green
fluorescent
protein-based bacterial biosensor for bioavailable toluene and related
compounds, Appl. Environ. Microbiol. 68:1962–1971.
• Southward, C. M., Surette, M. G., 2002, The dynamic microbe: green fluorescent
protein brings bacteria to light, Mol. Microbiol. 45:1191–1196.