Development Of A Biosensor Using Photobacterium
Spps. For The Detection Of Environmental
Pollutants
Ashvriya Thapa, Shweta Biswal, Dr Neeru Sood, Dr Ramchandran, Dr Trupti Gokale
Department of Biotechnology
Bits Pilani Dubai Campus
Dubai, United Arab Emirates
Abstract— Bioluminescence is a natural phenomenon exhibited
by many organisms, in which energy is spent to emit light. These
organisms emit light through an enzymatic reaction between the
substrate Luciferin, and an enzyme Luciferase. The Lux operon
controls the process of luminescence, which follows the quorum
sensing mechanism. We used the idea of bioluminescence to develop
a photobiosensor to detect the presence of environmental pollutants.
Photobacterium spps. was isolated from the squid and the effect of
environmental pollutants was studied in the organism by analyzing
the luminescence exhibited by the bacterium in presence and absence
of the pollutant. The luminescence was recorded using Perkin Elmer
Victor 320. A linear decrease in the luminescence was observed with
the gradual increase in the concentration of the tested heavy metals.
However, a surprising result was observed in presence of Barium (up
to concentration 0.6 mg/ml) where the luminescence was found to
increase and decreased with further increase in the concentration.
This signifies the ability of the bacterium to sense the presence of the
pollutant quantitatively as well as qualitatively. This luminescent
property of Photobacterium spps. can be exploited for the
development of a photobiosensor. This paper discusses the design of
an optical biosensor using fibre optic technique and photodiodes to
detect the luminescence and convert it into a measurable signal.
Keywords—Photobacterium;Bioluminiscence;Heavymetals;
Metallothionines; Photobiosensor.
I. INTRODUCTION
Photobacterium is a gram negative bacteria which belongs
to family of Vibrionaceae. It is found in symbiotic relationship
with sea organism like squid. It is a luminescent bacteria. In
presence of substrate luciferin, the enzyme luciferase oxidizes
luciferin to produce light [1]. Luminescence property of
Photobacteria can be used to develop a biosensor to detect
heavy metals in the environment.
One of the major problems faced today is soil
contamination. It occurs due to the accumulation of metalloids
and heavy metals, through emissions from rapidly expanding
industries, disposal of high metal wastes, mine tailings, paints,
fertilizers, pesticides, gasoline etc.
Heavy metals have exhibited several negative effects on human
health, for example chronic inhalation of cadmium is suspected
to cause lung cancer and progressive renal tubular dysfunction
[2]. Exposure to barium can lead to respiratory failure,
paralysis, elevated blood pressure and kidney damage.
Ingesting high concentrations of copper salts can cause nausea,
dizziness, vomiting, diarrhea and tachycardia. Gastrointestinal
effects (nausea, vomiting, and diarrhea), effects on the blood,
liver injury, and allergic dermatitis have also been reported in
humans from oral exposure to cobalt [3]. Hence, it is important
to detect the presence of these heavy metals in the environment
followed by their remediation. In this paper we propose to
develop an optical biosensor for the detection of heavy metals.
II. MATERIALS AND METHOD
A. Isolation of Photobacterium
Photobacterium spps. was isolated in our laboratory from
the squid using BOSS media and identified using the 16s rRNA
analysis. [1]
B. Effect of heavy metals on the luminescence of
Photobacteria
Five different heavy metals were selected for the study. Stock
solutions of the selected heavy metals were prepared as the
following; Cadmium chloride (20 mg/ml), Barium chloride
(30 mg/ml and 300 mg/ml), Zinc suplate (1 mg/ml), Cobalt
chloride (2.5 mg/ml), Copper sulphate (30 mg/ml). Varying
concentrations of the metal salts were made by diluting the
stock solutions. 24h old culture of Photobacterium grown in
Luminescent broth was exposed to working concentrations of
metals- 1mg/ml Cadmium, 150mg/ml Barium, 1mg/ml Zinc,
1.25 mg/ml Cobalt and 1.5mg/ml Copper. The effect of metals
on luminescence of Photobacterium was monitored up to
30hrs using Perkin Elmer Victor 320.
C. Overexpression of genes in presence of heavy metals
Effect of heavy metals on gene expression was studied using
Zinc sulphate, Silver nitrate, Barium chloride and Copper
sulphate. Gene expression in terms of protein analysis was
done by growing the Photobacterium for 24h in presence of
the four selected salts. Working concentrations for barium
chloride, copper sulphate and zinc sulphate was 20 mg/ml and
concentration of silver nitrate was 10 mg/ml. In a second
experimental setup, grown culture of Photobacterium (24h
old) was further incubated with the same concentrations of
heavy metals as mentioned above and immediately checked
for its protein analysis. SDS PAGE was performed to study
the protein profiles [4].
III. RESULTS AND DISCUSSIONS
A. Growth of Photobacteria
Photobacterium spps. was routinely grown in Luminescent
broth for 24h at 28C (Fig.1)
Figure 1: Photobacterium spp.grown in luminiscent broth for 24h.
B. Effect of heavy metals on the lumiscnece of Photobacteria
(a) (b)
(c) (d)
(e)
Figure2: Effect of Heavy metals on luminescence of Photobacterium spps.
a) Cadmium - Line equation of the graph is Y= -89535x
+244991. R=0.893756. The Error is = 1-R= 1-0.893756
=0.106.
b) Barium- Line equation of the graph is Y= -482.09x +
172585. R=0.990403. The Error is = 1-R= 1-0.990403=0.01.
c) Zinc- Line equation of the graph is Y= -4E +6X +809446.
R=0.973190. The Error is = 1-R= 1-0.973190 =0.02680.
d) Cobalt- Line equation of the graph is: Y= -78390X
+285864. R=0.917496. The Error is = 1-R= 1-0.917496.
e) Copper- Line equation of the graph is: Y= -10279X +
294731. R=0.9695. The Error is = 1-R= 1-0.96695 =0.033046
C. Overexpression of genes in presence of heavy metals
Figure 3 : SDS-Page gel containing protiens isolated from Photobacteria.
Effect of selected 5 metals was checked on the luminescence
of Photobacteria. Photobacterium was observed to be
sensitive to cadmium as its luminescence decreases linearly
with increasing concentration (Fig 2.a). Other metals such as
copper, zinc, and cobalt also exhibited a similar sensitivity.
However, in case of barium there was an unexpected linear
increase in luminescence up to 6 mg/ml of barium. Upon
further increase in the concentration, a drop in luminescence
was observed (Fig 2.b). A unique observation was the ability
of Photobacterium to emit luminescence in presence of a high
concentration of barium up to 150mg/ml. this suggests a low
sensitivity of Photobacterium towards barium.
Photobacterium was observed to be most sensitive to copper,
as seen from figure 2e.
The gene expressions of Photobacteria was seen in the
presence of different metals using two experimental set ups.
Observation of extra bands or enhanced expression was
observed for Zn, Cu and Ag in the first experimental setup.
This can suggest production of metallothionein. Observation
of extra band was seen in case of Zn in second experimental
setup. The cells grown/ exposed in presence of metals such as
Zn, Cu, and Ag exhibited extra protein bands as compared to
the control. This could be due to expression of some new
proteins in the presence of these metals. However, in presence
of Barium, there was no difference exhibited between the
control and the cells grown/ exposed to Ba.
The cells are found resistant to Ba. Thus it may have a
mechanism to resist the impact of this metal and due to this
the luminescence is not affected.
IV. DESIGN OF BIOSENSOR
A biosensor is an analytical device, used for the detection of
an analyte (pollutant), which combines a biological
component (Photobacteria) with a physicochemical detector
[5].The photo detectors can detect the intensity of the light
emitted by any object radiating photons. The output of the
photodiode is connected to the amplifier and intensity of the
light emitted can be recorded in terms of voltage .The voltage
Vout depends on the amount of light incident on the photo
device
Figure 4: Circuit Diagram of the Sensor
Figure 5: The Proposed Device of the Biosensor
Biosensors consists of a biological sensing element, which in
this case is the effect on luminescence produced during
reaction between Photobacterium and the heavy metal [6].
A transducer/detector which detects the light emitted by the
bacterium and converts it into an electrical signal. Usually
these are photodiodes or photoelectronic devices. They can
detect the luminescence and converts the light signal to an
electrical signal.
The light is measured counting the photons rather than flux.
These instruments can absorb low irradiance. They convert
these irradiance to 4-20 mA electrical signals. These signals
can be directly read as degree of luminescence or amount of
input.
Advantages of developing an optical biosensor using
Photobacterium spps. are real time analysis of an analyte
(sample suspected to contain heavy metal), it is precise and
highly sensitive, results obtained were reproducible and
without hysteresis. The device is small and compact and can
be made specific towards a heavy metal by introducing the lux
genes under a respective promoter for the gene.
V. CONCLUSION
Using Photobacterium as a biosensor, we can easily detect
heavy metals as its luminescence decreases in their presence.
This method is rapid and environment friendly.
From the experiment it is seen that the luminescence of the
bacteria decreases in presence of heavy metals. A gradual
decrease is observed as the concentration of the heavy metals
increased in the reaction mixture except in case of barium
where the luminescence increased up to concentration of 6
mg/ml. Photobacterium was observed to be highly sensitive to
copper, and exhibited a steep drop in the luminescence, while
least sensitive to barium.
The biosensor is designed using optical principles, making it
less expensive and easy for handling. This may prove to be a
promising feature.
ACKNOWLEDGMENT
We would like to express our heartfelt gratitude to Prof. R. N.
Saha, Director-Bits Pilani Dubai Campus, who has given us an
opportunity to apply and understand our engineering concepts
in a practical atmosphere.
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