INTRODUCTION:

Most of the organisms have a metabolic and their own biochemical pathways which are of actually the necessary factors for its survival. But in deep oceans where the living conditions are not only depended on these metabolic factors but also depends on a major physiological phenomenon which is known as Bioluminescence as the name suggests it is light produced by living organism. This is living light for living^1,4.

Cnidarians, Angler fish, lantern fish and certain fishes are bioluminescent but all of them are not self some are symbiotic association with Bacteria which are responsible for the light production

Indian mackerel is a common Indian fish which also exhibits bioluminescence as they have pass through the Phosphorescence Noctilusa Bacterium which attach to them appear luminescent^2.

Basic Principle and Biochemistry of Bacterial Bioluminescence:

Biochemistry of Bacterial Bioluminescence:

There is a special enzyme in the luminescent bacterial system which is known as the luciferase. This luciferase is an enzyme complex with two sub units as alpha unit and beta unit .These two sub units make it as a large unit as of nearly 80kda.

The bacterial bioluminescence is of proceeds in a pathway as the energy compound as FMNH2 is interacted with the O2 and along with long chain fatty aldehyde groups in the presence of bacterial luciferase. This results in formation of Fatty acid and water molecule, but the main point is the energy rich FMNH2 is converted to Flavin Mono nucleotide and remaining energy is released as light³.

Here we have to notice that though the both subunits are interdependent, if the Beta subunit is absent then alpha sub unit shows an inefficient function resulting in poor light yield. It is also be noted that besides the luciferase there are many important enzymes which are indirectly involved in this light production process as they are responsible for the formation of substrates for this continuous light emission.

Chemical Reaction (general)

FMNH₂ + O₂ in presence of Bacterial luciferase along with long chain Fatty-aldehyde results in FMN and Fatty acid by loss of H₂O.

Energy released as light.

Genes of bioluminescent-bacteria:

In luminescent system like bacteria there are special nucleic acid sequences which code for the proteins (enzymes) which are responsible for the action. These sequences are known as the lux genes and thus “lux A and lux B“codes for luciferase alpha sub unit and beta sub unit respectively. Besides this unit there are lux C, lux D and lux E which codes for the enzymes which helps for the substrates⁵

Photobiology of Bioluminescence:

The obtained (produced) light in bioluminescence is of calculated in Specific bioluminescence units or Spectral radiance ⁶

Blue Green light—PE =490nm =>> 0.06lux/O.D or Lu/area

Blue light---PE=478nm = >>0.02lux/O.D or Lu/area

Yellow ---PE=545nm = >>0.23lux/O.D or Lu/area

MATERIALS AND METHODS:

Sample Collection:

The selected species are collected from the Vellore (New Fish Market). The species include (Indian mackerel, Rohu, Black-spot Snapper, Fin bream and Indian Goat Fish).

The fishes are actually well cleaned (pre storing processing) then they are placed separately. By using (clean) synthetic gloves and in sterile bottles (used for samples) the fish scales are carefully collected. After collection the samples are taken for lab processing NOTE: At the time collection the (scales) fishes are in ambient temperature of 29 degree Celsius.

Laboratory Conditions:

The collected samples (scales) which are placed in sterile bottles are now transferred to cooling conditions i.e. they are placed in a refrigerator which is maintained at 4degree for controlling the bacteria and protection of samples from damaging⁷.

General Procedure:

Biochemical Characterization:

This included the steps as

(i) Media

(ii) Staining

(iii) Microscopic Observation

Media: Nutrient Agar Media is used

Morphological study by staining method:

Then before going for staining it must be noted that the entire proceeding experiments are done in aseptic/sterilized conditions.

SIMPLE STAINING:

Simple staining

From streaked media smear is taken on slide

Then methylene Blue is added as staining dye

Then left for 2 mins

Now slides are washed

And are observed in Microscope (pics taken)

Flow chart showing the procedure of simple staining.

GRAM STAINING:

i. Gram positive bacteria-violet

ii. Gram negative bacteria‐pink

PROCEDURE in brief

First the smear has been made

Then first Primary dye is added to it

Then it is washed with iodine

And now a counter stain is added

Different bacteria shows differ stains in observation (pics are taken)

ENDOSPORE STAINING:

First the smear has been made

Then first the slides are placed over a steam

Then we added the malachite green over steam on slides

Then it is left for 2mins and washed

Now we added saffranin as other dye

Bacteria produces spores under unfavour conditions shows green stains

Then microscopic observation (pics are taken)

Flow chart showing the pathway for the procedure of Endospore staining.

Thus from the obtained colonies of bacteria their biochemical characterization is done.

These tests and results are shown in the table.

Growth kinetics studies for isolated strain:

Growth phases at different types were determined. Graph between (O.D value) Vs time was plotted. Bacterial growth in batch culture can be modeled with four different phases:

(a) In the lag phase, the bacteria get to use with the available conditions for growth and hence they get mature and doesn’t get divide

(b) Exponential phase (Log phase) is the next phase of bacterial growth in this phase the cells divide and divide to increase the number of cells that means in this phase multiplication occurs. This can be shown by plotting number of cells vs time producing a straight line as graph for this mode of growth that occurs exponentially.

(c) Then the next phase is the stationary phase where the available nutrients for bacteria gets reduced thus the rapid growth slowed

(d) As bacteria attains the death phase, they get deficit of nutrients and die. At this phase, nutrients limited and toxic substances are increased so as a result the death cells are more than growing cells.

The graph of different phases of growth of bacterial cells are shown in the figure^8.

Tolerance Assay:

Bile salt tolerance:

Imperviousness to bile salts is viewed as an imperative parameter for selecting probiotic strains. A convergence of 0.2, 0.4, 0.6 % of bile salt has been suggested as an appropriate focus for selecting probiotic microorganisms for human utilize. So we have to decide the bile salt resistance with various grouping of bile salt in the media for that overnight culture (1 % v/v) was hatched in MRS juices containing 0.2, 0.3, 0.4 (w/v) oxgall (brondicahispanlan, SH).PH of control and test were changed in accordance with 6 with 1N HCl or NaOH and was brooded vigorously for 6 hour. Culture turbidity was hourly measured.

NaCl tolerance:

The NaCl tolerance of isolated lactobacillus is determined by as 10 test tubes containing MRS broth were taken and adjusted with different concentration level (8%) of NaCl. Sterilization aws done and then, each test tube was incubated with 1 % (volume/volume) in fresh that one-night culture of lactobacillus is of made and at 37⁰C for 24-hour incubation is done. After 24 hour of incubation the turbidity was observed which is used in determination. By variation in growth level ++ for maximum and + for normal and – for non growth.

Acid tolerance:

MRS agar broth for incubation of organisms. Then pH of MRS broth was adjusted in different values as of 2.0, 3.0 and 4.0 with 1N HCl and at pH of control was adjusted as pH 6. In this one night the cultures were inoculated (10% vol/vol) and incubated at 37°C for 3 h and growth was monitored using the plate count method. Samples are taken on MRS and then plates at 24hr for 37°C incubation is done .So thus tolerance was detected ⁹.

Cholesterol reduction:

For every culture to be tried, 70 μl of cholesterol arrangement was added to 10ml of MRS juices (last cholesterol fixation 70 μg/ml). To the MRS soup, 1% of newly developed culture was included and hatched at 37°C for 24 hour. A uninoculated test was utilized as control. After brooding the cells were evacuated by centrifuge about 10 mins at 10000 rpm (4 degree) and cholesterol was resolved in the supernatant utilizing adjusted Rudel and Morris (1973) technique where the 3 ml of obtained supernatant and 2 ml of 33% (wt/vol) KOH solution and 3 ml of 96% ethanol form were blended and stirred for 20 seconds and hatched for 15 min at 60°C in a water shower. After hatching, the blend was cooled under faucet water, then 5 ml of hexane and 3 ml of water were included and vortexed for 1 min. Tubes were permitted to remain for 15 min at 30ºC to allow stage detachment. Then first 2.5 ml of hexane layer taken and dried and next 1.5 ml of Ferric chloride reagent was taken and rest of 10 min is given then 1 ml of concentrated sulphuric acid is added. This entire setup us blended and left for 45 in at 30 degree temperature. The optical thickness was measured at 570 nm in UV spectrophotometer and centralization of cholesterol was resolved.

(Conc. of cholesterol in control) – (Conc. of cholesterol in sample)*100

Assimilation (%) =--------------------------------------------------------------------------

(Conc. of cholesterol in control)

RESULTS AND DISCUSSION:

Based on the data (obtained) from the different staining techniques we have seen that the different scales of species of fishes as Indian mackerel, Rohu, Black-spot Snapper, Fin bream & Indian Goat Fish different types of gram positive bacteria which also produces spores on unfavorable conditions. This all results also shows that these bacteria are predominately lives on scales of this fishes. We can now also observe from the biochemical test the type of reactions that these obtained bacteria (can) show different types of the results [products]. It is to be noted that from the dermal scales of this fishes we have successfully obtained the different types of bacteria that are present on them. It is also to be in discussion that these scales (dermal) are taken or collected from the (local) VELLORE fish market that means, these are actually from edible fishes as we know that the Rohu, Black-spot Snapper, Fin bream etc are more frequently are of consumed.

CONCLUSIONS:

The observable results are 10X of sample 3 in Fig4, 40X of sample 4 in Fig4 and 10X of sample 4 in Fig6 Showing the luminous shots i.e from Indian mackerel and Black spot snapper (species of Phototrabhdus and Noctilusa).

Thus it is an important aspect that these fishes which indirectly are of also association with these bacteria. Now from this staining and microscopic observations which also leading to biochemical tests also conclude that these bacteria are present. As we know that bioluminescent bacteria are also capable of producing toxins they are to be controlled. Thus by these steps we can determine them.

ACKNOWLEDGEMENT:

We sincerely like to express our gratitude to Dr. G. Viswanathan, founder and hon’ble Chancellor, VIT University, Vellore, for his constant support and encouragement. We want to express special thanks to Dr. Sekar Viswanathan, Mr. Sankar Viswanathan and Mr. G.V. Selvam for their constant motivation and help and we want to thank DST and MTCC for financial assistance and microbial cultures.

Table 1 shows the reported bioluminescent Microorganisms (Bacteria and Fungi)

Bacteria	Habitat	Distribution	luminous	Color of light	Photon energy level	Scientist
Photobacterium	Marine	South and east	Yes	blue	478nm	Thomas Poinar(1979)
Vibrio fisheri	Marine	Phillipines	Yes	yellow	545nm	Beijerik and Lehnmann (1896)
Noctilusa	Marine/ fresh	Indian	Yes	Blink white	Not clear	Zeebruge(1886)
Photorhabdus	Marine	Indo-Pacific	Yes	Blue green	490nm	Martinus(1880)

Table 2 shows collected sample and their data

S. No.	Common Name/local name	Scientific name	Habitat	Distribution
1	Rohu	Catlacatla	Marine	Bay of Bengal
2	Indian goat Fish	Purprneusindicus	Marine	West coast
3	Indian Mackerel	Rastrelliger	Marine	Indian ocean
4	Black spot snapper	Latjanus	Fresh/Marine	West and east of india
5	Indian fin bream fish	Nemipterushexodon	Marine	Indian ocean

Table 3 shows the agar media components.

Ingredient	Composition
Starch	2gm
Peptone	0.5g
Beef extract	0.3g
Distilled water	100 Ml

Table 4 Shows the Various Biochemical Tests

Test		Sh	Sh	Sh	Sh
Indol		-	-	-	-
Catalase test		-	-	-	-
Citrate utilization test		+	+	+	+
Starch hydrolysis test		+	+	+	+
Fermentation of carbohydrate test	Glucose	+	+	+	+
	Sucrose	+	+	+	+
	Lactose	+	+	+	+
Mannitol test		-	-	-	-
Methyl red test		+	+	+	+


Rohu Black spot		Snapper

Fin bream fish	Indian goat fish	Indian Mackerel