Toxicity Biosensor for the Evaluation of Cadmium Toxicity Based on Photosynthetic Behavior of Cyanobacteria Anabaena torulosa
Wong Ling Shing,
Lee Yook Heng
A biosensor based on electrochemical transduction using an oxygen probe
has been developed for the measurement of Cd2+ toxicity by
using cyanobacteria (Anabaena torulosa), where the cells were immobilized
on the surface of an oxygen probe. The biosensor responded to the changes
in photosynthetic oxygen release under illumination by a light source.
Exposure to Cd2+ at concentrations below approximately 8 mg
L-1 did not demonstrate any inhibition but stimulation of oxygen
production was observed. Inhibition occurred only when concentration of
Cd2+ was above 8 mg L-1. Using the same concentration
range of Cd2+ but increasing the exposure time of Cd2+
from 15 to 30 min, inhibition of oxygen release was mainly observed.
This pattern of inhibition of A. torulosa by Cd2+ was
different from that of Cu2+, which was also measured by the
same toxicity biosensor, where no stimulation in oxygen release was observed.
The use of toxicity biosensor has enabled the detail toxicity behaviour
of A. torulosa towards Cd2+ to be evaluated.
Whole cell based biosensors can respond to a wide range of changes in
their environment and suitable for use in toxicity test and environmental
monitoring where the sources and nature of the toxicant cannot be predicted
(Bentley et al., 2001). The biosensors response to real physiological
impact of active compounds present in the samples and act as broad band
monitoring for toxins, which might not necessary discriminate between
the different types of toxicants (Giardi et al., 2001; Mattiasson
1997; Roger, 1995; Carpentier et al., 1991; Evans et al.,
1986). Biosensors can be configured to be sensitive and inexpensive to
manufacture and can be significant in terms of reducing cost and increase
the efficiency of certain environmental monitoring applications (Roger,
Anabaena torulosa is a type of filametous cyanobacteria (cyanophyceae)
from Nostocaceae family, which formally known as blue-green algae (Linda,
2000). The organism contains chlorophyll a and undergoes photosynthesis
and the physiology changes can be observed by oxygen evolution. Based
on the research by Chay et al. (2005), the organism has shown potential
as a good biosensor to copper (Cu), lead (Pb) and 2,4-Dichlorophenoxyacetic
acid (2,4-D). However, the previous studies had not investigated the usefulness
of cyanobacteria A. torulosa as a biosensor for Cd toxicity evaluation.
In view of Cd is a toxicant, which is non-essential, non-beneficial and
possesses high toxic potential (Giardi et al., 2001; Lockwood,
1976) and a common pollutant in water resources (Heever and Grobbelaar,
1998), the focus of this study is to assess the usefulness of A. torulosa
as a biosensor for rapid Cd toxicity evaluation.
MATERIALS AND METHODS
Copper (2) nitrate, Cu(NO3)2, Cadmium (2) nitrate,
Cd(NO3)2 (Merck, Germany), poli-hydroxylethyl methacrylate,
(pHEMA) (Sigma UK), 1,4-dioxane (Fisher, UK), cyanobacteria Anabaena
torulosa (Carolina Biological Supply Co., US), Bold`s Basic Medium
(James, 1979) as culture medium for Anabaena torulosa. All reagents
were prepared in distilled water.
A. torulosa Culture
A. torulosa was cultured in Bold`s Basic Medium at 18.5Â°C,
1000 Watt unit-1 area white fluorescent illumination, with
light and dark period maintained at 16 and 8 h, respectively in culture
chamber (GC-500, Protech). Each liter of Bold`s Basic Medium prepared
contains 10 mL of several macronutrients stocks and 1 mL of trace elements
stocks respectively (Table 1). Aeration was carried out by manual shaking
three times daily to avoid cells clumping.
Determination of Cells Growth
The number of cells was determined by using a Microscope BX51 (Olympus,
USA) and a Weber haemocytometer. Concentration of suspended cell can be
estimated by using UV/VIS spectrophotometer (Perkin Elmer). The optical
density at 700 nm can be used to estimate the cell density for immobilization
as the two quantities were closely related. OD at 700 nm is the absorption
peak of chlorophyll a in A. torulosa (Desikachary, 1959). The growth
of the cyanobacteria was determined every 2 days for 14 days to establish
the growth profile and cells at the most active phase of the growth were
selected for toxicity biosensor studies (Chay et al., 2005).
||The preparation of stocks for Bold`s Basic Medium
The pHEMA solution was prepared by dissolving the polymer in a mixture
of water and dioxane with the ratio 4:1 and 1 mL of solution contained
60 Î¼g of pHEMA. By using 50 Î¼L of pHEMA solution, a certain
amount of A. torulosa cells were coated on a Teflon gas permeable
membrane. The cyanobacteria entrapped in the pHEMA film were left to dry
at 18.5Â°C for 4-5 h. The membrane was then assembled onto the tip
of an oxygen electrode (Orion, USA) by using a rubber O-ring. The oxygen
evaluation can be measured by a change of dissolved oxygen when the immobilized
cells were illuminated by a 100 W lamp (Phillip Classictone, Holland)
compared to non-illuminated condition. The change of oxygen level was
recorded using a PC (DO sensorlink PCM 800, Thermo Orion, USA).
Exposure of A. torulosa to Various Concentrations of Cadmium
Cd toxicity tests were conducted for both immobilized and free cells
of A. torulosa. The amount of pHEMA used for immobilization, cell
density and cell culture age were optimized. Each biosensor was used only
once for toxicity evaluation at each concentration of Cd. The experiment
for immobilized cyanobacteria was performed in pH 7 and at room temperature,
as stated by Chay et al. (2005). The biosensor was left in 10 mL
of distilled water for 15 min until a stable baseline was obtained. The
photosynthetic oxygen release of the immobilized A. torulosa cells
was determined continuously for 5 min after light illumination began.
The biosensor probe was then incubated in 10 mL of various concentrations
of Cd2+ (0.1, 0.25, 0.5, 1.0, 5.0, 10.0, 20.0 mg L-1)
solutions for 15 min at room temperature. After the period, the probe
was then transferred to 10 mL of distilled water again and illuminated
to allow photosynthetic activity for another 5 min. Any inhibition of
the immobilized cells on the oxygen probe can be calculated as follows:
||Highest amount of oxygen evolved before exposure to the Cd ions
within a fixed duration.
||Highest amount of oxygen evolved after exposure to the Cd ions within
a fixed duration.
|The highest amount of oxygen
|| The highest oxygen value observed within a fixed duration
- the baseline oxygen value
In free cell (i.e., non-immobilized cells) toxicity evaluation for A.
, 10 mL A. torulosa
cells with an absorbance unit of
1.0 Abs at 700 nm was left for 15 min until a stable baseline was obtained.
Further illumination for 5 to 10 min was performed to oxygen evolution.
Various amount (<0.1 mL) of 100 Î¼g L -1
of Cd solution
was the added to the free cell solution to make the total Cd concentration
range from 0.1-5.0 Î¼g L-1
. It was then incubated for 15
min. Illumination was conducted once again at 5 to 10 min to determine the
oxygen evaluation. The percentage of photosynthetic inhibition was then
RESULTS AND DISCUSSION
Immobilization techniques in biosensor offer advantages in terms of controlling
the number of cells, better cell stability, more reproducible response,
enabling continuous monitoring and greater mechanical strength (Mattiasson,
1997; Trevan and Mak, 1998). The usage of a thin layer of gel to immobilize
the cells provides a fast diffusion of gases and short analysis time (Philp
et al., 2003).
Cd toxicity tests on immobilized A. torulosa used in the biosensor
construction showed that at lower concentrations of Cd, i.e., below approximately
8 mg L-1 of Cd there was no inhibition on photosynthetic oxygen
release. Beyond this value, the inhibition of oxygen production occurred
(Fig. 1). In fact, stimulation of oxygen release was observed at low concentration
of Cd and the response is varied as the oxygen release can increase by
up to 100% in one instance and only < 20% in another.
In photosynthetic organisms, Cd2+ can affect several metabolic
activities in different ways. Some examples are photosynthesis and growth
inhibition, binding onto chlorophylls, chlorosis and decrease in nutrient
and water uptake (Clijsters and Assche, 1985; Krupa, 1999; Nies, 1999;
Prasad and Strzalka, 1999, Clemens et al., 2002). Several studies
in isolated chloroplasts have confirmed that Cd2+ affected
photosynthesis in both the PS 1 and PS 2 (Atal et al., 1991; Siedlecka
and Krupa, 1996; Pagliano et al., 2006).
A. torulosa is a photosynthetic organism. Although Cd can inhibit
the activities of PS 1 and PS 2, results from this study showed that A.
torulosa has mechanism to mediate the effect of Cd2+ especially
at low concentration and after a short period of exposure. The mechanism
appeared to accelerate the photosynthetic rate and produced more oxygen.
The damage caused by low level of Cd2+ might initiate some
biochemical processes that are related to the self-recovering mechanism
from damage. The cyanobacteria cells used in the experiments were from
the most active phase of growth and hence they may be capable of influence
the Cd2+ transformation process in the cells (Krishnan et
However, the inhibition of photosynthesis began as soon as the Cd2+
concentration increased beyond the threshold value where the recovering
mechanism no longer operational. Similar behaviour was also observed for
the non-immobilized cells but in this case inhibition was observed until
1 mg L-1 of Cd after which stimulation occurred. At 5 mg L-1,
the cells demonstrated inhibition again (Fig. 2).
The more susceptible of the non-immobilized cells to inhibition by Cd
when compared with the immobilized cells is presumably attributed to the
immobilized cells being protected from the pHEMA matrix that used to immobilize
the organisms. This enables the organism to launch its self-recovery mechanism
when Cd2+ is present.
The exposure duration of Cd2+ can have different effects on
the toxicity behaviour. At short duration of exposure to Cd2+,
an organism can adapt to higher Cd2+ concentrations by employing
resistance mechanisms, such as expression of Cd2+-sequestering
compounds (e.g., phytochelatins) or exporters capable of transporting
Cd2+ out of cell (Nies, 1999). It has been observed that the
cyanobacteria Trichodesmium erythraeum bloom when the Cd2+
concentration increased to 0.31 mg L-1 (Krishnan et al.,
2007). For A. torulosa studied here, The IC50 for 30
min of exposure is 0.9 mg L-1. However, the IC50 for
15 min of exposure was not reached even with very high concentration of
Cd2+ (Fig. 3).
||The response of the toxicity biosensor containing immobilized A.
torulosa when exposed to various concentrations of Cd2+.
Expt 1 and Expt 2 are duplicate experiments
||The response of non-immobilized (free) A. torulosa
cells to various concentrations of Cd2+ demonstrate a response
similar to the immobilized A. torulosa
||The effect of the length of exposure time on the inhibition behaviour
of A. torulosa by Cd2+
||The inhibition pattern from various concentrations of Cu2+
on the toxicity biosensor containing immobilized A. torulosa.
The reproducibility of the biosensor to Cu is indicated by the errors
on the data
The inhibition of oxygen observed throughout the Cd2+
used still demonstrated the cells attempted to recover after an initial
inhibition by Cd2+
during the prolonged exposure to Cd2+
to 30 min.
The peculiar behaviour of Cd2+ toxicity on A. torulosa
did not exhibited by other metal such as copper (Cu). Exposure of the
biosensor to Cu2+ using the present experimental setup showed
no increase of oxygen production by the cyanobacteria (Fig. 4). In fact
inhibition of oxygen production was observed throughout when exposed to
Cu2+and the response of the biosensor was linear with the concentrations
of Cu2+. This is in agreement with previous finding by Chay
et al. (2005) who used a same experimental setup to study Cu toxicity
of A. torulosa.
In other species of algae or cyanobacteria, the presence of Cd2+
induced a significant decrease in activities of both PS 1 and PS 2, which
led to inhibition of oxygen production. The level of inhibition on oxygen
release yielded IC50 value of 5-10 mg L-1 in Chlamydomonas
sp. and for Anabaena inaequalis, 1 mg L-1 of Cd2+
significantly inhibited the photosynthetic rate (Nagel and Voigt, 1995;
Stratton and Corke, 1979). For Anabaena flos-aquae, the LD50
96 h for Cd2+ was reported to be 0.14 mg L-1 (Heng
et al., 2004). Thus, even Anabaena species demonstrated different
toxicity response to Cd2+. Based on the results observed here,
some further studies may be useful to understand how the behaviour of
this cyanobacteria species when under Cd2+ exposure.
The toxicity biosensor for the evaluation of Cd2+ toxicity
on A. torulosa demonstrated that the Cd2+ toxicity
behaviour of the cyanobacteria is peculiar where at lower concentration
of Cd2+ (<8 mg L-1), stimulation of the photosynthetic
activities occurred. This is in contrast to metal such Cu2+ toxicity
on the same cyanobacteria where the stimulation phase was not observed
even at much lower concentrations Cu2+ when compared to that
of Cd2+. However, when the exposure time of the organism to
Cd2+ was increased to 30 min, the biosensor registered a diminished
effect of the stimulation phase and the inhibition effect became dominant.
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