Isolation and Identification of Bioluminescent Bacterium from Marine Fishes - Toxicity of Metals Assessed by Effect of Metals on Growth of the Bacterium

Home , Bioluminescent bacteria

R. Lakshmi1, N. Arunagirinathan2*

1Dept of Microbiology, Justice Basheer Ahmed Sayeed College for Women, Chennai- 18. 2PG and Research Department of Microbiology and Biotechnology, Presidency College, Chennai- 5.

Abstract: Bioluminescence is the process of visible light emission that occurs via exergonic chemical reactions. It occurs in living organisms, and the organisms that emit light are said to be bioluminescent. The light produced is a metabolic byproduct of the oxidation of organic compounds (Luciferin) facilitated by the enzyme Luciferase. Bioluminescent bacteria primarily inhabit the ocean either as free-living species or associated with other marine organisms in a symbiotic, parasitic or saprophytic relationship. Luminous bacteria can be used successfully to determine toxicity of microquantity of different substances: heavy metals, phenols, narcotics, toxins, pesticides, medicines etc. Fifty samples of ocean fishes were processed to isolate and identify bioluminescent bacteria. 4 metals powders were used to assess the toxicity of the metals based on their effect on the growth of the bacterium which was studied from optical density (OD) determination and plate counts.

Key words: Bioluminescence, Luciferin, Luciferase.

Introduction Bioluminescence is the production and emission of light by a living organism as the result of a chemical reaction during which chemical energy is converted to light energy (1, 2). Luminous

*Author for Correspondence. E-mail:[email protected] 12 LAKSHMI AND ARUNAGIRINATHAN bacteria are a convenient object to be used in solving general biological problems, genetic and genetic-engineering researches. Strains from the luminous bacterium collections are used as a basis for preparation of Lyophilized bacteria for measurements of the integral biological toxicity of different substances (3). In the last 30 years, the discoveries in bioluminescence have revolutionized the area of environmental microbiology, but have also shed light on the area of ecological, industrial and medical significance (4, 5).

Mechanism of Bioluminescence Bioluminescence involves the combination of two types of substances in a light-producing reaction. One is a luciferin, a light-producing substance. The other is a luciferase, a heat-labile enzyme that catalyzes the reaction. The luciferin is a heat-stable protein known as photoprotein. Neurological, mechanical, chemical or as-yet discovered triggers can start the reactions (6). Bioluminescence reactions require the presence of molecular oxygen and other substrates like Adenosine triphosphate (ATP). In the bacterial Luciferase-catalysed reaction, the energy expenditure on the reduction of molecular oxygen to a peroxy reaction intermediate and then ultimately to water serves as a trigger for releasing the potential energy from the oxidation of both FMNH2 and fatty aldehyde in the form of photon emission (7, 8).

Materials and Methods Sample Samples of fresh ocean fishes were obtained from Marine beach, Chennai and examined for the presence of bioluminescent bacteria.

Collection and Transport The fresh dead fishes were purchased from the markets near the Marina sea coast. They were then placed in a plastic bag with trapped air and laid on a crushed ice for storage and brought immediately to the laboratory. The purpose was to protect any growing colonies from smearing.

Processing the Sample The fishes were first washed slightly with sterile distilled water to remove the mud or sand particles. The dead fish was then laid into sea water half submerged for about 24-48 hours at 18- 22oC, or refrigerated at 4-8o C for inoculation.

Media required for Cultivation Luminescence medium (LM), Luminescent broth, Luminescent Agar (LA), BOSS medium were used. All the cultures were well aerated, so as to support sufficient amount of oxygen needed in luminescence reaction. On solid medium, the glowing could be observed in 1-5 days after inoculation, but faint glowing could be observed longer (even few weeks).

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Methodology Isolation of bioluminescent bacteria A lawn culture was made initially from the fish on to the Luminescent media using aseptic techniques. The plates were examined every 4 to 6 hours for growth and were observed for luminescence in a dark room. A loopful of luminescent colonies were inoculated on to sterile media plates of luminescent agar, luminescent medium and boss medium using sterile inoculation loops. Bioluminescence was looked for in a dark room at regular intervals (9, 10).

Presumptive identification bacteria Gram staining, motility, catalase, oxidase were done for the isolates and further characterized by biochemical and sugar fermentation tests.

Media used for identification Thiosulphate Citrate Bile salt Agar (TCBS), MacConkey Agar.

Toxicity Effects of Metals on Growth of Bioluminescent Bacteria 4 metal powders were used, namely, Zinc, Ferrous, Copper and Aluminium, to test their effect on growth of the bioluminescent bacterium.

Procedure Metal stock solutions were made in 2% NaCl solutions obtained from osmotic adjusting solution (22% or 22gms of NaCl in sterile distilled water). The culture was inoculated from medium into 7ml of liquid NaCl based medium using a sterile loop and were incubated overnight under still conditions in glass tubes at room temperature. The culture was then subsampled and diluted 1000 (10-3) times into another 7ml of NaCl based medium and gently agitated on a reciprocal shaker at room temperature. Under these culture conditions, the lag phase lasted about 20 hours after inoculation, followed by a log phase of about 30 hours, ending in a stationary phase approximately 55 hours after inoculation. The bioluminescence per cell changed during this time, increasing in early log phase, peaking at mid- log phase, and decreasing during late log phase, as previously reported (Hasting & Nealson,1977)(11,12). Based on this characterization, the experimental window in this study was restricted to a 9-hour period (from 24 to 33 hour after inoculation into liquid NaCl based medium) to ensure a constant growth rate and an increase in bioluminescence. The metal treatment involved the addition of 1000 microlitres of the metal stock to the bacterial culture. Beginning from 24 hour after the liquid inoculation, cell concentration was measured at times 0 hour, 1/2, 1, 2, 3,4,6,9 hours, after a 3s vortex mixing to allow homologous sampling. Cell concentration was determined from optical density (OD) measured with spectrophotometer at 620nm. After each experiment, the culture was tested for possible microbial contamination by inoculating the

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Results The study was made to isolate the bioluminescent bacteria from marine fishes and to observe the effects of metals on the growth of the bacterium. 50 samples of ocean fishes of 4 varieties were examined for the presence of bioluminescent bacteria.

Isolation of Bioluminescent bacteria Macroscopic Observation White, pinpoint, regular shaped colonies were observed suggesting the presence of Vibrio phosphoreum, since the colonies of Vibrio fischeri are large, yellow pigmented. The temperature at which the culture showed optimum growth and bioluminescence was at about 4-6O C. These results showed that it is Vibrio phosphoreum, unlike Vibrio fischeri which grows at 30 - 35o C.

Microscopic Observation Presumptive identification of bacteria Gram negative rods were seen on gram staining, actively motile, catalase positive and oxidase positive.

Identification of bacteria using selective media 1. TCBS (Thiosulphate Citrate Bile salt Agar) - Yellow pigmented colonies were observed indicating the presence of Vibrio species. 2. MacConkey Agar - Large circular, non-lactose fermenting colonies were observed. Biochemical tests and carbohydrate fermentation tests were performed. Amino acid decarboxylase test is the confirmation test for identifying the species (Table 1).

Table 1. Amino acid Decarboxylase Test

Sl. No. Amino Acid Initial Stage Middle Stage Final Stage ( 24 (0 Hour) (12-18 Hours) Hours or Longer) 1. Lysine Purple Yellow Yellow 2. Arginine Purple Yellow Yellow 3. Ornithine Purple Yellow Yellow The above results confirm Vibrio phosphoreum, since for Vibrio fischeri, Ornithine is negative.

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Effect of Metals on Growth of Bioluminescent Bacterium The effects of metal powders on growth of bioluminescent bacteria were assessed. Growth was determined from optical density (OD), measured with spectrophotometer at 620nm. All the metals were found to affect the growth. Copper was the most toxic of the 4 metals viz. Zinc, Ferrous, Aluminium and Copper, as indicated by the results of OD determination (Table 2 and Figure 1).

Table 2. Showing the Effect of Metals on Growth - OD Determination by Spectrophotometry Time(Hrs) 0 0.5 1 2 3 4 6 9 Control 0.05 0.09 0.13 0.15 0.17 0.25 0.31 0.39 Zn 0.05 0.08 0.1 0.13 0.15 0.18 0.21 0.24 Cu 0.03 0.043 0.051 0.072 0.09 0.13 0.15 0.16 Fe 0.04 0.07 0.09 0.12 0.16 0.18 0.21 0.25 Al 0.07 0.09 0.13 0.15 0.17 0.21 0.25 0.29

0.5 Figure 1. Effect of Metals on Growth 0.4

0.3 Control 0.2 Zn Concentration(OD)

0.1 Cu Cell 0 Fe 0 0.5 123469 Al

Time

The OD value of the culture incorporated with copper was reduced more in contrast to other metals, indicating that copper was the most toxic of the 4 metals viz- zinc, ferrous, aluminium and copper.

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Discussion Wegrzyn and Czyz, 2002, suggested the potential biotechnological applications of bioluminescent marine bacteria, mainly in the detection of mutagenic and toxic compounds in marine environments. In the present study the effect of metals on the growth and toxic effect on the bacterium. The results also correspond with those of Van Der Lelie.D. et al., 1994. Copper plays an important role in regulation of growth and bioluminescence in Vibrio phosphoreum. This element maintains a delicate balance between promoting growth, which has a stimulating effect on light production, repressing bioluminescence (13).

Conclusion Remarkable opportunities are offered by the use of luminous bacteria in the various areas of biology. Their ecological and technological applications indicate that they deserve close attention of biologists and other experts of the adjacent areas of knowledge. Because of its successes in nature, humans are beginning to understand the significance bioluminescence can have in research queries. Assays based on animals and plants are expensive, time consuming and require large sample volume. Our study emphasizes the benefits of rapid reproducible and cost-effective bacterial assay for toxicity, screening and assessment.

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9. Baumann, P., Baumann, L. and Mandel 1971. Taxonomy of marine bacteria. Bacteriology 107: 269-294. 10. Parvez, Z., Venkatraman, C., Mukerji, S. 2001. Advantages of implementing luminescence inhibition test for acute toxicity prediction of chemicals. P: 42-49. 11. Nealson, K.H. and Hastings, J.W. Bacterial bioluminescence: Its control and ecological significance. 1989. P: 496-518. 12. Van Der Lelie, D., Corbisier, P., Baeyens, W., Wuertz, S., Diels, L. and Mergeay, M. 1994. The use of biosensors for environmental monitoring. 145: 67-74. 13. Nealson, K.H. and Hastings, J.W. 1992. Luminous Bacteria. In: Balows, A., Truper, H.G., Dworkin, M., Harder, W. and Schleifer, K.H. (Eds). The Prokaryotes, a Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Application. 2nd Edition New York.Springer-Verlag, 1:625-639.

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