Environment and Natural Resources J. Vol 8, No.

3, December 2010:1-9 1

The Potential of Luminescent Bacteria ‘Photobacterium leiognathi’ as a Biosensor for

the Detection of Aquatic Toxicity

Beh Weng Chee*, Lim Yee Kheng, Asmat Ahmad, Lee Yook Heng and Salmijah Surif
Faculty of Science & Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Malaysia

Abstract

Evaluation of environmental aquatic toxicity is a convenient way for environmental

pollution management before any detailed chemical pollutant analysis is performed. In this
study, the luminescent bacteria Photobacterium leiognathi was used to evaluate the toxicity
polychlorinated biphenyl (PCB) and lead (Pb). PCB at 2 ppm to 10 ppm and Pb, 0.001 ppm
to 100 ppm were exposed to bacteria and the resulting bioluminescence was measured
spectrophotomatically at 484.74 nm. There were consistent decreases in bioluminescence
with increased toxicity of Pb. However, the bioluminescence was inhibited and did not
show consistent decrease with increasing concentration of PCB. Photobacterium leiognathi
appear to be selectively responding to toxicants at certain level of concentration.
Photobacterium leiognathi bacterium is potentially useful as biosensors especially for Pb
but not for the PCB. However, more work needed to be carried out to determine the detail
responses pattern and the threshold value for Photobacterium leiognathi in respond to Pb

Key words: Bacteria/ Bioluminescence/ Luminescent/ Photobacterium leiognathi/ Toxicity

1. Introduction which can cause disruption of the

metabolism (Girotti et al., 2008). In aquatic
Environmental monitoring of environments, bacteria play an important
pollutants is becoming increasingly role in metabolite activities and nutrients
important to the general public. This is cycles (Gellert et al., 1998). A living
particularly true for compounds that pose a microbe is a package containing all the
potential risk to human health or to the substances necessary for complex chemical
environment (Sung et al., 2000). In recent reaction without the needs of any external
years, bioassay studies on toxicants using substances which makes a living microbe a
bacteria as a sensing organism have been sensitive detector to its surroundings
promoted due to their sensitivity, ease to (Tauriainen’ et al., 2000).
use, cost-effective and fast responses for The bioluminescence or chemo
detection and evaluation of environmental luminescence is an aerobic bio-oxidation
toxicity (Froehner et al., 1999; Kudrysheva, process alternative to normal bacterial
2005; Tencaliec et al., 2005; Girotti et al., respiration (Perego et al., 2002). It is a
2008). Bioassay measures changes in characteristic shown by a number of
physiology or behavior of living organisms marine organisms and land animals
resulting from stresses included by including bacteria (Rees et al., 1998;
biological or chemical toxic compounds, Girotti et al., 2008) of about 666 genus
* Corresponding author.
E-mail address: salmij@[Link]
2 Beh W. C. et al. / Research Article: 1-9

from 13 phyla (Girotti et al., 2008). Photobacterium phosphoreum, Photo-

Besides bacteria, bioluminescence is also bacterium leiognathi, and Alteromonas
exhibited from phyla as low as hanedai (Ainul, 2002). Luminescence in
dinoflagellate to marine vertebrates, not Photobacterium leiognathi and other
higher than fish (Girotti et al., 2008). luminuous bacteria is the product of bacterial
Bioluminescent assay generally luciferase, a mixed function oxidase that uses
shows good correlation with other toxicity oxygen, reduced flavin mononucleotide, and
bioassays such as algae, crustacean, and a long-chain fatty aldehyde as substrates to
fish (Girotti et al., 2008). The most produce blue-green luminescence (Ast et
thoroughly studied bioluminescent al., 2007).
bacterium used in toxicity testing is The bioluminescent enzyme system
Photobacterium phosphoreum (also known contains oxidoreductase NAD (P): FMN,
as Vibrio fischeri), a marine bacterial strain and component of luciferase which emits
(Frymier and Ren, 2002). A commonly lights in the presence of FMN (flavin),
used test method that employs P. NAD (P) H, a long chain aldehyde (RCHO)
phosporeum is the Microtox test (Frymier and oxygen molecules (Kudrysheva, 2005;
and Ren, 2002). Bioassay based on bacteria Hernando et al., 2007; Girotti et al., 2008).
is a gaining popularity as useful method for Bacteria’s luciferase is very specific to
an early warning system for environmental FMNH2 (mononucleotide flavin), and at the
monitoring and pollution management due same time shows a weak activity to the
to its sensitivity to toxic chemical others flavins (Girotti et al., 2008).The
substances and the fast results that can be metabolise energy produced in this pathway
obtained from such assay. (Girotti et al., is changed to chemical energy through the
2008). electron transport system to visible light
Luminescent bacteria are from (Hernando et al., 2007) as in equation (1)
Vibrionaceae family (Tai, 2004; Medvedeva et and (2) (Girotti et al., 2008). In equation
al., 2005) and there are 3 genera namely (2), with the presence of luciferase, a
Photobacterium, Vibrio, and Alteromonas species of heterogen enzyme, light is
(Tai, 2004; Frischer, 2005). A few produced as a by products in the catalysis
examples of luminescent bacteria are Vibrio reaction (Tauriainen’ et al., 2000).
fischeri, Vibrio harveyi, Vibrio logei,
oxidoreductase
NAD(P)H + FMN + H NAD(P)+ + FMNH2 (1)

FMNH2 + RCHO + O2 luciferase FMN + RCOOH + H2O + Light (2)

In this work, the species of bacteria 2. Material Studied

P. leiognathi was investigated as a
potential biosensor for environmental The luminescent bacteria, P. leiognathi
toxicant, PCB and Pb is investigated. was isolated from squid and supplied by
Toxicity test is based on inhibition of School of Biosciences and Biotechnology of
light production by the luminescent Universiti Kebangsaan Malaysia (PPBsBt,
bacteria (El-Alawi et al., 2000). UKM). P. leiognathi is a marine
bioluminescent bacterium that is widely
distributed in tropical and temperate coastal
environments, and it is also known as a light
 Environment and Natural Resources J. Vol 8, No.3, December 2010:1-9 3

organ symbiont of fish species belonging 3.4 Determination of Optimal Bio-

to the families Leiognathidae, luminescence Measurement
Acropomatidae, and Apogonidae
(Orndorff and Colwell, 1980; Wada et al., 3.4.1 Wavelength
2006). P. leiognathi is chosen due to its Bacterial suspension of 3.0 ml was
strong luminescence which can be pipetted into a 4 ml of 4 clear sides cuvette
detected easily. and their luminescence scanned at
wavelength from 200 nm to 800 nm by using
3. Methods and Techniques a fluorescence spectrophotometer (slit
opening: 5 nm; scan speed: 150 nm/minute)
3.1 Growth Medium (Perkin Elmer LS55). Excitation light source
was fixed at 0 to eliminate any background
Medium for growth of bacteria fluorescence. The data was processed by FL
(OXOID) was prepared by adding 28 g of Winlab software.
nutrient agar and 20g (2%) of sodium
chloride (NaCl) to 1000 ml of distilled 3.4.2 Optimal Time for using bacterial
water and heated until completely suspension
dissolved. The nutrient agar contained The optimum time for culturing
Lab-Lemco flour (1g L-1), yeast extract (2 luminescent bacteria is 18 hours (Girotti et al.,
g L-1), peptone (5 g L-1), NaCl) (5 g L-1) 2008). The bioluminescent intensity was
and agar (15 g L-1). It was autoclaved at measured every 30 minutes for 6 hours to test
1210C for 15 minutes. The solution was its intensity stability. The first bioluminescent
poured into Petri dishes and stored in cool intensity was measured half hour after the
room. optimum cultured time

3.2 Cell Culture 3.4.3 Determination of Cells Density

A serial of bacterial suspensions (3:0;
All manipulations were done in 2:1; 1:1; 0.5:1; 0.5:5; 0:3)v/v in saline
aseptic conditions. Individual colonies solution were prepared in triplicates. A serial
were transferred into the Petri dish. The mixture of 0.9 µL was dropped between a
bacterium was incubated for 18 hours at Weber hemacytometer and a slide. The
30oC (Girotti et al., 2008). number of bacterial cells was counted with
BX51 microscope (Olympus, USA) equipped
3.3 Growth of Bacterial Suspension with 100x magnification. The light intensity
of the mixture was then measured by
Saline solution was used in this fluorescence spectrophotometer.
study to reduce noise in spectro-
photometer. [Link], a marine 3.5 Toxicants Preparation
species need 2% NaCl (saline solution)
for suspension (Zhang et al., 2008). Polychlorinated biphenyls solutions
Bacteria from Petri dish were rinsed twice (PCB No. 52) (Dr. Ehrenstorfer) were
with 30 ml of saline solution into a beaker prepared by dissolving with 1% methanol
to produce bacterial suspension and was (J.T Baker) before diluting to 2, 4, 6, 8, and
transferred into a conical flask and shaken 10 ppm using distilled water. The solutions
well (250 rpm) at room temperature. were stored at 4oC until use and is stable for 3
4 Beh W. C. et al. / Research Article: 1-9

months. Lead (Pb) solutions (0.001, 0.1, 1, where, Io: saline solution with bacterial
5, 10, 30, 50, 80, and 100 ppm) were suspension
prepared from standard lead nitrate ABN, It: toxicants with bacterial suspension
Australia solution (Pb in 0.5% of nitric
acid) and are stable for 1 year. 4. Results and Discussion

3.6 Toxicity Test 4.1 Peak Emission Wavelength of

Photobacterium leiogntahi
A total of 1.5 ml of toxicants (PCB
and Pb) was added to 1.5 ml of bacteria [Link] was shown to emit light at
suspension in a cuvette containing 266 ± 484.74 nm (Figure 1) maximally and this
20 of cells bacteria. The mixture was wavelength was used throughout this study.
incubated for 30 minutes and the cuvette The wavelength has been used consistently in
was covered with parafilm to prevent this study. This emission is slightly lower
contamination from surroundings. For than another bioluminescent that has been
control, 1.5 ml of saline solution was used in many bioassay studies, [Link]
added to 1.5 ml of bacteria suspension. with the reported wavelength of 490 nm
The bioluminescence inhibition was (Frymier and Ren, 2002; Nagata and Zhou,
calculated based as the equation below 2005; Girotti et al., 2008). Different species
(Wong et al., 2008). has been showed to emit bioluminescent at
different wavelength (Haddock et al., 2010).

134.2

120

100

80

I nt Int.60

(a.u)40

20

0.0
200.0 400 600 800 900.5
nm

Figure 1: The wavelength of luminescent bacteria

4.2 Optimal Time the highest intensity that is within the 6 hours
after the optimum culturing time to ensure
Within 6 hours the experiment was good signal of emission.
undertaken, bioluminescence intensity
remained relatively constant with only 4.3 Density of Cells
small variation of +8 units. In this study,
fresh luminescent bacteria assay was used Figure 2 shows the relation of
within the optimum time range and bioluminescence intensity at different
conditions where bioluminescence shows numbers number of bacterial cells.
 Environment and Natural Resources J. Vol 8, No.3, December 2010:1-9 5

Figure 2: Bioluminescence intensity and number of bacterial cells

Bioluminescence intensity showed a sensing, depend on cell population density

linear relationship to the bacterial cells (Strauss, 1997; Holloway, 2004; Tai, 2004;
(Pearson correlation p<0.01, R2=0.9674). Stolper et al., 2008).
The bacteria bioluminescent are
controlled by autoinducer, a signal which 4.4 Toxicity Testing
is accumulated during cell growth and
detected by receptor which in turn can Figure 3 showed bacterial response to
excite the luciferase enzyme. It will cause Pb. This experiment was repeated and the
the luminescent reaction when the results showed increase bioluminescence
concentration achieves threshold value inhibition pattern with increase toxicity. The
(Katznelson and Ulitzur, 1977; Strauss, readings for bioluminescence inhibition were
1997; Tai, 2004). Coordinating in the range of 4.59% to 58.03%.
mechanism function and accumulation of
auto-inducer also known as quorum

Exp. 1 Exp. 2

Figure 3: Bioluminescence inhibition pattern cause by exposure to Pb.

The response of the [Link] to test shows the significant correlation (p<0.01)
an inorganic pollutant, Pb is shown at between bioluminescence inhibition and
Figure 4. Two ways Pearson Correlation increased Pb toxicity. Girotti et al. (2002),
6 Beh W. C. et al. / Research Article: 1-9

reports that as the toxicants concentration and both sets of data shows regular pattern
increase, the light emitted by but did not showed continuous
bioluminescent bacteria will decreased. bioluminescence inhibition. The results
This indicates that the cell energy status showed bio-luminescence inhibition at 2, 4, 8
has been disturbed or cell structure was and 10 ppm. However, the results showed
damaged (Backhaus et al., 1997) after the decrease in bioluminescence inhibition at 6
bacterial cells are incubated with ppm for both sets of data. The
toxicants. Although there is no study has bioluminescence inhibited in the range of
been conducted on [Link] before, 36.69% to 57.06%. At the concentration
Tsiridis et al. (2005) reported the almost tested, bioluminescence was inhibited
same inhibition profile by Pb with V. moderately 2 to 10 ppm. It is not clear why
fisheri. concentration at 6 ppm was lower in the
Figure 4 shows bacterial response bioluminescence inhibition.
with PCB. The experiment was repeated

Exp. 1 Exp. 2

Figure 4: Bioluminescence inhibition pattern cause by exposure to PCB

Steevens et al. (1999) reported no [Link] although pesticide concentration

significant bioluminescence inhibition for exists at ppb level.
concentrations higher than 24 ppm and Not all organic toxicants can inhibit
below 1.6 ppm for anthracene and bioluminescence by bacteria. From previous
benzo[a]pyrene because both toxicants are studies, nalidixic acid shows no effect on
relatively insoluble in water (<45 ppb). inhibition though the incubation period with
Therefore bioavailability of the 2 PAHs the bioluminescent cell was increased
may significantly influence the exposure (Backhaus, 1997). For Choi and Gu (2001),
and effect on bioluminescence inhibition. their studies do not show obvious
Farré et al. (2000) showed that bioluminescent respond for two out of five
chlorfenvinphos at 0.2 ppb and 0.3 ppb and tested toxicants, which included 2, 4, 5-
on endosulfan between 0.007 ppb and 0.12 trichlorophenol (2, 4, 5-TCP) and
ppb does not show any toxicity effect to pentachlorophenol (PCP).
 Environment and Natural Resources J. Vol 8, No.3, December 2010:1-9 7

Bacterial cell sensitivity to various Universti Kebangsaan Malaysia.

toxicants has a threshold value. The range Unpublished.
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degradation (Tamar and Schaefer, 2001). comparison between the long term (24
The presence of ions such as the h) and the short term (30 min)
saline (2% NaCl) as used in this study may bioassay. Chemosphere 35 (12):
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