BIOLUMENESCENCE

Biology research project

Name: surya prakaash | class: 11th | division: science
NITTE INTERNATIONAL SCHOOL
Content
1. Introduction
2. Biochemical events of light emission

3. Range and variety of bioluminescence

4. Application of bioluminescence in
biotechnology and beyond
5. Experiment
6. bibliography

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Introduction
You may have seen the sparkle of fireflies on a summer’s
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 given off in the
process. For that reason, the emission is called cold light
or luminescence.

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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.

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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,
hatchetfish, Apogon, and Parapriaeanthus.

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 FIG: chemical mechanism of bioluminescence in firefly

In firefly luminescence, the substance adenosine

triphosphate (ATP) initially reacts with firefly
luciferase, ionic magnesium, and firefly luciferin
to form a 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
(FMNH2). In the complete reaction, bacterial
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luciferase reacts with FMNH2 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.

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 FIG: CHEMICAL REACTION OF BIOLUMENESCENCE 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.

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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 situ in complex food
systems, for problem-solving and for the development of
modified and improved processing and storage purposes as
discussed further below

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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.

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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, fibre 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.

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EXPERIMENT
AIM: TO OBSERVE THE BIOLUMENESCENCE IN A
DINOFLAGELLATES
PROCEDURE
 At any time: View cells under microscope.
 To be performed in a darkened room during the night phase of the
dinoflagellates.
o Mechanical stimulation: rap with knuckles, swirl around, or bubble air in
flask containing culture.
o Chemical stimulation: add some drops of 10% acetic acid (or vinegar) to a
test tube containing part of your culture. The increased acidity of the
solution activates the luminescent chemistry within the cells.
 Pour cells gently into small dish and place under microscope. Add a
few drops of 10% acetic acid (or vinegar) while observing/measuring
luminescence.
 The effect of light and dark cycle on dinoflagellate bioluminescence.
o Grow some dinoflagellates on a normal day/night cycle, while others are
grown on a reverse cycle so that they have their nighttime during our day.
o Stimulate (shake) the two batches and observe/measure which produces
brighter bioluminescence.
o NOTE: In most dinoflagellates, bioluminescence is minimal during their
day.
 The effect of light inhibition on dinoflagellate bioluminescence.
o Take dinoflagellates during their night cycle and expose half to room
lights while the others are kept in the dark.
o After 30 minutes in each condition, stimulate (shake) the two batches and
observe/measure which produces brighter bioluminescence.
o NOTE: Nighttime dinoflagellate bioluminescence is inhibited by light
exposure.
 The effect of illumination on the brightness of dinoflagellate bioluminescence.
o Grow batches of dinoflagellates at different light levels during the day.
This is done by varying the distance of the batches from a light source.
For example, double the distance means one quarter the light intensity.
o During the dark phase, stimulate (shake) the batches and
observe/measure the brightness of bioluminescence.
o NOTE: Less illumination can mean less energy for the dinoflagellates.
 The effect of stimulus strength on the brightness of dinoflagellate
bioluminescence.
o Place batches in dinoflagellates in vertical columns, e.g., graduated
cylinders.

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 o Use an aquarium air pump with a control valve that is adjusted to have
different air streams, as tested in a dish/tank using tap water. Use tubing
and air stones so they reach into the graduated cylinder.
o During the dark phase, turn on the air pump and observe/measure
the brightness of bioluminescence as a function of the amount of bubbling
from the air stone. Bubbles really stimulate dinoflagellate
bioluminescence!

BIBLOGRAPHY
i) Wikipedia
ii) Britannica
iii) National geographic.org
iv) Latz laboratory
v) Pubs.rsc.org
vi) Encyclopedia

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