No. 12, Myllappanahalli, Yelahanka, Bengaluru North-560089.

(Affiliated to CBSE Code: 830332)

CBSE CLASS – 12

SUBJECT
INVESTIGATORY PROJECT
(2022-2023)

TOPIC: BIOLUMENESCENCE

SUBMITTED TO: Mrs. MAMATHA

SUBMITTED BY: SURYA PRAKAASH
CLASS : XI SCIENCE
REG. No. : 27
 CERTIFICATE
This is to certify that ______________________________________ of class 12 has satisfactorily
completed his/her Project Report in ____________________on the topic _______________
_______________________________________ as prescribed by the Central Board of Secondary Education
for the partial fulfillment of AISSCE 2022 – 23.

Date:

Signatures:

Internal Examiner: External Examiner:

Principal: School Seal:

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Introduction
You may have seen the sparkle of fireflies on a summer night.
The fireflies produce light through a chemical reaction in their
glowing abdomens, a process known as bioluminescence. But
did you know that seascapes can also glow and glitter thanks
to the light producing abilities of many marine organisms?
Some fish dangle a lighted lure in front of their mouths to
attract prey, while some squid shoot out bioluminescent
liquid, instead of ink, to confuse their predators. Worms and
tiny crustaceans also use bioluminescence to attract mates.

Bioluminescence is the emission of light by an organism or by

a laboratory biochemical system derived from an organism. It
could be the ghostly glow of bacteria on decaying meat or fish,
the shimmering radiance of protozoans in tropical seas, or the
flickering signals of fireflies. The phenomenon occurs
sporadically in a wide

Range of protIsts and animals, from bacteria and fungi to

insects, marine invertebrates, and fish, but it is not known to
exist naturally in true plants or in amphibians, reptiles, birds,
or mammals.

Bioluminescence results from a chemical reaction

(chemiluminescence) in which the conversion of chemical
energy to radiant energy is direct and virtually 100 percent
efficient; i.e., very little heat is give off in the process. For that
reason, the emission is called cold light or luminescence.
Darwin was not the first to note bioluminescence. Greek
Philosopher Aristotle observed that bioluminescence is a type
of "cold" light - in that it does not produce heat - in around
350 BC. Researchers have since found that this form of
chemiluminescence, produces blue-green light as a result of
the oxidation of a compound called luciferin (the "light-
bringer) by an enzyme called luciferase.
Biochemical events of light emission

In most bioluminescent organisms, the essential light-

emitting components are the Oxidizable organic molecule
luciferin and the enzyme luciferase, which are specific for
different organisms. The present custom is to Use generic
names according to origin

e.g., Firefly luciferin and luciferase, Vargula luciferin and

luciferase. The luciferin-luciferase reaction is actually an
enzyme-substrate reaction in which luciferin, the substrate, is
Oxidized by molecular oxygen, the reaction being catalyzed by
the enzyme luciferase, with the consequent emission of light.
The light emission continues until all the luciferin is oxidized.
That type of reaction is found in fireflies, Vargula, Latia, and
many types of fish, such as lantern fish, hatchet fish, Apogon,
and Parapriaeanthus
. In firefly luminescence, the substance adenosine
triphosphate (ATP) initially reacts with firefly luciferase, ionic
magnesium, and firefly luciferin to forma complex (luciferase-
luciferyl- adenylate) and pyrophosphate. That complex then
reacts with molecular oxygen to emit light. Enough energy is
liberated in the last step to convert the electronic
configuration of the luciferase-luciferyl-adenylate complex
from a low-energy ground state to a high-energy excited
state. The high-energy complex then loses energy by radiating
a photon of visible light and returns to the ground state.

Luminescent bacteria employ the enzymatic Oxidation of

reduced Flavin mononucleotide (FMNH). In the complete
reaction, bacterial
Luciferase reacts with FMNH, and oxygen to form a long-lived
intermediate complex, which then reacts with a long-chain
aliphatic aldehyde molecule (e.g., decanal) to emit light.

THE RANGE AND VARIETY OF

BIOLUMENESCENCE

Luminous species are widely scattered taxonomically, with no

discernible pattern. Many luminous shrimps are known but no
luminous crabs. Many luminous squids are known but only a
single luminous octopus (Callistoctopus arakawai of Japan).
Again, luminous centipedes and millipedes are not
uncommon, but luminous scorpions and spiders are
apparently nonexistent.

Almost half the animal phyla contain luminous forms, but the
number of representatives is very small compared with the
total number of known animal species. The protists are not so
rich in luminous species but are greatest in sheer abundance,
especially in tropical seas, In fact. the majority of luminous
organisms are marine.
 Fig: chemical reaction of bioluminescence in bacteria

The ocean surface in many parts of the tropics is dense with

single-celled luminous planktonic organisms, primarily
dinoflagellates that glow when stimulated mechanically, as by
the churning of the waves, or, when washed ashore, by the
pressure of a foot. Some organisms exhibit a 24-hour rhythm
of light intensity, highest at night and lowest during the day.

Among crustaceans, luminous species are especially

remarkable in the copepods, shrimps, and ostracods.
Applications of bioluminescence in biotechnology
and beyond

Hygiene control
Bioluminescence based sensing technology has been of great
use in hygiene control for several decades now. In particular
the ATP-bioluminescence assay based on the firefly
bioluminescence system is routinely used to monitor the
cleanliness of surfaces in healthcare facilities such as
hospitals and clinics and in the dairy and meat processing
industries. This is the technique of choice when the speed and
ease of analysis are of vital importance as alternative
methods such as culturing or microorganisms usually take
days to offer results, whilst techniques based on fluorescence
need an external light source for excitation and do' not
discriminate between living and dead cells. Bacterial
bioluminescence is also used in the food industry to monitor
the behavior of Lux-tagged bacteria in sSitu in complex fOod
systems, for problem-solving and for the development of
modified and improved processing and storage purposes as
discussed further below
Mapping pollution in ecosystems

The most widely used bioluminescence sensors in the

Toxicology monitoring of ecosystems are whole-cell bacterial
bioluminescence sensors Like most bioluminescence-based
assays, these sensors provide a quick result to help assess
toxicity levels. Other protocols for measuring environmental
toxicity often involve exposing test organisms, such as fish,
crustaceans, plants or bacteria to environmental samples and
to monitor survivorship. The benefit of bioluminescent
bacteria is that their light output can be used as a quick
measure of survival. Moreover, their bioluminescence is
directly linked to their respiratory chain and so any toxin that
interferes with their respiratory chain, interferes with the light
Output. These sensors have been used to monitor a wide
variety of contaminants including both heavy metal
Contaminants and organic compounds such as toluene and
naphthalene.
Culture and heritage- preservation of art work

The ATP bioluminescence assay from the firefly has also been
adapted to take surface measurements of ATP from old
artwork to estimate bacterial, fungal, yeast, algae and lichen
levels in antique work for the purposes of cultural and
historic preservation. A bioluminescence low-light imaging
technique was reported that was used on artwork consisting
of paper, stone, fiber and wood. All reagents were applied
directly to the samples and the conditions were optimized for
Sample geometry and surface conditions. More recently, the
level of the microbial contamination of the seventeenth-

century wall paintings in the nave of the old Church of the

Holy Ascension (Veliki Krcimir, Serbia) was evaluated using
the ATP bioluminescence method, and traditional cultivation-
based method, using dip slides that were commercially
available. It was established that ATP bioluminescence
measurements can be a quick way to determine 'hot spots' of
contamination on the art-work, allowing a quick assessment
of areas that require greater concern.
Experiment
Aim: to create the bioluminescence found in firefly
FIREFLY BIOLUMINESCENCE

Preparations

Firefly Buffer. Dissolve 0.8 g of glycine and 0.25 g of ammonium

bicarbonate in 200 ml of distilled water. Adjust the pH to about 7.6
with dilute HCl or NaOH.

ATP Solution. Dissolve 0.1 g of adenosine triphosphate (ATP) and

0.05 g MgCl2 in 100 ml of firefly buffer.

1. Extract the bioluminescence system in vitro. Fireflies are

common during the summer months in the Americas and Asia. If
they are so available, then collect 10-20 specimens (a butterfly
net works well) and allow them to dry thoroughly by whatever
means available, freeze-dry, desiccate, but avoid any heat. Cut off
the tails where the yellow light organ is obvious, and make a
"cold extract" of the tails by grinding with about 1 g of clean sand
in 3 mL of cold firefly buffer. A mortar and pestle is handy for
this, and keep everything cold by working in a cold room or by
surrounding the mortar with ice. Pipette the extract into a handy
container (e.g., test tube) and keep cold. Discard the particulate
left-over by filtration or by using a lab centrifuge. If collecting
insects in the field is not practical, dried firefly tails may be
purchased from several laboratory supply companies. Keep the
cold extract ice cold.
Now make a "hot extract" by repeating the grinding with fresh
tails in hot, not boiling, firefly buffer. Cool the test tube containing
the extract again in ice.

2. To "see" an enzyme substrate reaction at work. In the dark

room measure out about 1 mL of cold extract, and allow this and
the hot extract to come to room temperature (RT), or nearly so to
your touch. You will notice a low level of yellow light coming from
the RT-cold extract sample, which will soon die away. Add the
RT-hot extract quickly (with a syringe or Pasteur pipette) to the 1
mL RT-cold extract, and you will get a flash of light. This
experiment is an example of a luciferase (enzyme) luciferin
(substrate) reaction first reported and named in the late 19th
Century by R. DuBois, but in 1947, McElroy showed that the hot
extract was in fact ATP, not firefly luciferin.

3. Assay of adenosine triphosphate. Take another 1 mL of cold

extract and bring to room temperature, and squirt in 1 mL of ATP
solution, and you will see the same flash of yellow light.

4. Bioluminescence color shift. To the final 1 mL of room

temperature cold extract, add several drops of 10% acetic acid.
Then immediately add another 1 mL of ATP. The light will now be a
dull red. It is hard to get the amount of acetic acid added to give
just the right acidity, so if this does not work, instead of the acetic
acid, add 1 drop of 10% zinc chloride, mix well, followed by the
ATP. This will also produce the red bioluminescence.
BIBLOGRAPHY

1. wikipedia.org
2. britannica.com
3. National geographic.org
4. Ncert class 11 biology text book
5. Pubs.rsc.org
6. Photobiology.info
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