The Effect of Light Treatment and Media Combination on Luminescence Endurance of Bioluminescent Bacteria Isolated from Squid [ Loligo duvacelli ( D ’ Orbigny , 1835 ) ]

Some bacteria emits light in the dark. The aim of this research is to find lighting duration and medium composition which produce the most enduring luminescence. The results showed that one of the bacteria isolated from light organ of Loligo duvacelli (D’Orbigny, 1835) squid are able to emit light. The isolation use trisalt solution and cultured into nutrient agar with addition of NaCl emits the longest blue-green light duration under a 1 : 1 dark-light shift incubation 8.3 d. Meanwhile under a total light, total dark, and in the oven incubator it illuminate for 4.2 d, 3.6 d and 2.6 d, respectively. The combination of beef extract + peptone + a commercial agar + NaCl generate the longest duration of luminescence when incubate in the oven incubator (3,2 d). Meanwhile it illuminated for 2.6 d and 1.7 d on NA + NaCl and NC + NaCl media, respectively, but it failed to illuminate on PCA, NA, and green beans extract + commercial agar + NaCl media. Based on this research we conclude that a commercial agar is potential to replace a technical bacterial agar function.


Introduction
Bioluminescence is a chemical reaction that takes place in an organism and produces detectable light.These organisms use a variety of body parts to emit light in different colors and for different purposes.This chemical process is different from fluorescence, another process that can cause things to emit light.In a few organisms, bioluminescence and fluorescence both occur [1].
Bioluminescence typically requires at least three components: a light-emitting organic molecule known as a luciferin; a source of oxygen (may be O 2 , but could also be hydrogen peroxide or a similar compound); and a protein catalyst known as luciferase.In some organisms, these three components are bound together in a complex called photoprotein.Light production may be triggered by the presence of ICBS Conference Proceedings ions (often calcium) or other chemicals.Some bioluminescent systems also contain a fluorescent protein that absorbs the light energy produced by the photoprotein, and re-emit this energy as light at longer wavelength.Several different luciferins have been found in marine organisms, suggesting that bioluminescence may have evolved many times in the sea among different taxonomic groups.Despite these differences, most marine bioluminescence is green to blue in color.These colors travel farther through seawater than warmer colors.In fact, most marine organisms are sensitive only to blue light [2].
Squid in Indonesia are known to emit light, helping the squid to find for food in the dark water, as well as a tool disguise from predators.Light emitted by a squid is due to the symbiotic relationship between squid species Loligo duvaucelli with bacteria Photobacterium phosphoreum that live in it [3].Luinescent bacteria light transmission is catalyzed by an enzyme called luciferase.The enzyme catalyzes the three substrates which reduced flavinmononucleotide (FMNH 2 ), oxygen (O 2 ) and long-chain aldehyde (RCOH).That reaction frees flavin (FMN), long chain fatty acids (RCOOH), and water (H 2 O) while emitting visible light [4].The aim of this research is to find lighting duration and medium composition which produce the most enduring luminescence.

Source
Squid [Loligo duvacelli (D'Orbigny, 1835)] were obtained from traditional markets in Malang, East Java.Ink organ of squid were separated from the body and crushed using mortar and pistil until homogeneous.

Medium
Technical medium was made by diluting 20 g NA instant, 30 g NaCl and 3 mL glycerol up to 1 × 10 3 mL of aquadest.NA liquid (LN) medium was made by diluting 3 g beef extract and 5 g peptone up to 1 × 10 3 mL of aquadest.DOI 10.18502/kls.v3i4.695

Addition of green beans extract, substitution of beef extract
About 1 kg of green beans were washed with water, peels are removed, then dried in the open air and boiled with a ratio of 1 × 10 3 g green beans: 2 × 10 3 mL of water, blended, filtered and reboiled.8 g of commercial agar (plain) were added in 20% of green bean extract and 3 g NaCl up to 1 × 10 3 mL aquadest.

Sterilization
All mediums were sterilized using an autoclave for 15 min at 121 ∘ C/15 PSI temperature (1 pascal is equal to 0.000145037738007 PSI, or 0.001 kPa).

Light provision treatment
The experiment was conducted in Microbiology Laboratory, State University of Malang.
Dark treatments were done by putting the culture in a closet and light treatment was done by placing the cultures in a constantly lighted room.Dark-light shift incubations were done by putting the cultured bacteria in a glass in front of the room as it followed the day-cycle.

Lighting duration under different medium treatments
The combination of beef extract + peptone + a commercial agar + NaCl showed the longest lighting duration of bacteria when incubated in the incubator (3.2 d).It illuminated for 2.6 d and 1.7 d on NA + NaCl and NC + NaCl media, respectively, but it did not illuminate on PCA, NA, and green bean extract + commercial agar + NaCl media.The data was analyze using SPSS 16 as shown in Table 4.
The results indicate that a significance of anova is 0.000 smaller than count significance 0.05.It means that type of medium affect the lighting duration of the bacteria.
From the statement, the test were continued using Duncan test to determine which

Conclusions
From the study we conclude that dark-light shift treatment is the most effective way to extend the lighting duration.A commercial agar is potential to replace a technical bacterial agar function.It is proved from the data that the replacement show the ability to grow the bacteria even a bit longer than technical agar usage.
and Peer-review under the responsibility of the ICBS Conference Committee.

Figure 1 :
Figure 1: The average of Lighting duration under provision light treatment.

2 : 3 : 4 :
in homogeneous subsets are displayed.a. Uses Harmonic Mean Sample Size = 10,000.T Analysis of light teatment duration using SPSS 16.TreatmentsLighting Duration (d)PCA NA NA + NaCl LN + NaCl Beef extract + Pepton + Lighting duration under different medium treatments (incubated 37 ∘ C).Analysis of lighting duration of different medium treatment using SPSS 16.

Figure 2 :T 5 :
Figure 2: Average value of lighting duration during different medium treatments.

Table 1 and
Figure 1 showed the highest to lowest average lighting duration in the Dark-

Table 2 .
Results showed that incubation treatment was not significantly different from dark room treatment but significantly different from light room treatment, light room treatments significantly different from the dark-light shift room treatment.Therefore, darklight shift treatments is the most effective treatment for the longest lighting duration of the bacteria.

695 Treatments Lighting Duration (d)* Incubator Dark Room Light Room Dark-Light Shift Room
*: Data were taken starting from the day after inoculation.