Abstract: In recent years the use of microbial sensors has widely applied for monitoring environmental contamination. In this study, we focus on the effects of biocides such as heavy metals, pesticides and herbicides on bioluminescent bacterium, vibrio fischeri strain DSM 7744 which is used as stable bioindicators. This method makes a correlation between the light of Vibrio fischeri and the concentration of biocides. However, the basic part of this research depends on how to optimize the best condition for maximum bioluminescence. Optimized conditions of Vibrio fischeri were stirring at 120 rpm at a incubation temperature within the range of 23 to 26°C after 24 to 48 h when solid cultures were reserved at 18°C. In this case we use the whole bacteria, Vibrio fischeri which is one of interesting bioluminescence bacteria, coupled with luminometer. In our procedure the LOD for two pesticides, Malathion and Diazinon, and two heavy metals, Mercury and Selenium is about 1ppb.
INTRODUCTION
The natural phenomenon of bioluminescence is the emission of visible light by living organisms mediated by an enzyme-catalyzed (luciferase) reaction of molecular oxygen with a substrate (luciferin) (Iwasaka and Ueno, 1998; Wilson and Hastings, 1998).
The use of bioluminescent bacteria as bioindicators dates back to the 1950s. There are variety examples of applications range using bioluminescence bacteria in assessment of environmental toxic components (Steinberg et al., 1995; Halldorson and Duran, 2003; Fenske et al., 2006). As bioluminescence bacteria are specially modified to respond to toxic concentrations of heavy metals by increasing an easily detectable signal, for example luminescence, they are very promising tools to detect bioavailable heavy metals such as Cd, As, Sb, Cr, Cu, Hg, Zn and Pb (Kahru et al., 2008). Conditions for bioluminescence of Vibrio fischeri in continuous culture has previously demonstrated (Scheerer et al., 2006).
Whole organisms are used to measure the potential biological impact (toxicity) of a water or soil sample. These systems are based on the use of luminescent bacteria, Vibrio fischeri, to measure toxicity from environmental samples. Bacterial bioluminescence has proved to be a convenient measure of cellular metabolism and consequently, a reliable sensor for measuring the presence of toxic chemicals in aquatic samples (Leitgib et al., 2007).
The bacterium V. fischeri is a well-described marine bacterium, which has a world-wide distribution and can be found preferentially in temperate and sub-tropical waters. It may grow in a free-living planktonic state or in a symbiotic relationship with certain fish and squid (Scheerer et al., 2006). In V. fischeri, there are two substrates, luciferin, which is a reduced Flavin Mononucleotide (FMNH2) and a long chain (7-16 carbons) fatty aldehyde (RCHO), which in its natural form is believed to be a tetradecanal. An external reductant acts via flavin mono-oxygenase oxidoreductase to catalyse the reduction of Flavin Mononucleotide (FMN) to FMNH2. The reduced flavin (FMNH2) binds to the enzyme and reacts with O2 to form a 4a-peroxy-flavin intermediate. This complex oxidizes the aldehyde to form the corresponding acid (RCOOH) and a highly stable luciferase-hydroxyflavin intermediate in its excited state, which decays slowly to its ground state emitting blue-green light with a maximum intensity at about 490 nm (Hastings and Nealson, 1977; Karatani and Hastings, 1993).
A biosensor is an analytical device that combines a biological sensing element with a transducer to produce a signal proportional to the analyze concentration (Lei et al., 2006).
Biosensors have been extensively applied in clinical, food and environmental areas due to the advantages of fast detection speed, high selectivity and sensitivity (Malhotra and Chaubey, 2003). In this study, the contribution of bioluminescence and luminometer makes a biosensor for detection of water pollution.
MATERIALS AND METHODS
Organism
The study was carried out at the Department of Chemical Engineering-Biotechnology,
Islamic Azad University, Science and Research Branch during 2006-2008. Vibrio
fischeri strain DSM 7744 was kindly provided by Iranian Research Organization
for Science and Technology (IROST).
Biociceds
Two organophosphorus pesticides: Diazinon and Malathion, two toxic metals:
Mercury (Ag2+) and Selenium (Se4+) are used in this experiment
HgCl2 and SeO2 were prepared from MERK®.
Diazinon and Malathion were purchased from Parto Nar Company.
Luminosity Measurement of Bacteria
A Berth old detection system (SIRIUS tube luminometer) made in Germany was
used to measure bioluminescence intensity.
Nutrient Media
To ensure the best quality of luminescent bacteria with sustainable viability,
the bacteria can be inoculated and maintained in culture medium. Although, a
variety of media mixtures can be used, the following cultures medium permit
maximum luminescence, growth and stability that are useful for the disclosed
methods.
Three basic growth media were tasted: 1. Bacto Marine Broth (DIFCO 2216) (Table 1) 2. Sea water agar (twin pack) (Table 2) (Fig. 1) 3. Sea water agar (Table 3).
The first media was used for reviving; the second one was used for solid cultures and the third one for liquid cultures.
| Fig. 1: | Vibrio fischeri in sea water agar (Twin Pack) media |
| Table 1: | Nutrient media used for reviving bacteria (DSMZ Medium 514:Bacto Marine Broth (Difco 2216)) |
| Table 2: | Nutrient media used for solid cultures. Sea water agar (Twin pack) |
| Table 3: | Nutrient media used for liquid cultures (DSMZ Medium 246: Sea Water Agar) |
Optimized Condition for Bacteria Growth
Solid cultures were reserved in incubator at 18°C. After 48 h liquid
cultures were incubated at 25°C in an orbital shaker at 120 rpm after inoculation
with brightly glowing V. fischeri solid culture (Claudia
et al., 2003).
Optimized Conditions for Experiments
Decrease of 500 μL bacterial luminescence in effect of 10 μL biocide
measured by luminometer after 2 min.
RESULTS
The luminescence of bacteria has long been known to be sensitive to a wide variety of toxic substances (eg., heavy metals, pesticides, etc.)for example the use of Luminescent bacteria has been discussed for the detection of toxins on solid surfaces, such as soil, and in liquid substances, such as in the analysis of waste water (Becvar and Becvar, 2002; Ulitzur et al., 2002).
The effect of four dangerous biocides (Mercury, Selenium, Malathion and Diazinon) in potable water was tested. Maximum light intensity were reached within 48 h after inoculation.Vibrio fischeri liquid cultures prepared from solid cultures and stirred at 120 rpm (Stanley and Kricka, 2002) for 48 h. Bioluminescence of 500 μL Vibrio fischeri was measured with luminometer when it was infected with each 10 μL of biocide.
Diazinon
Diazinon 60% at concentration of 100, 20, 10, 1, 0.01, 0.001 ppm were the
samples. Bacteria light in effect of Diazinon at 0.001ppm reduced 96% in comparison
of maximum bioluminescence.
It can be clearly seen that the bacteria light reduced in effect of Diazinon 60% at 100 ppm (about 97%).
There is not a great deal of difference between the reduction of light in effect of Diazinon at 100 and 0.001 ppm. But the most important point is an extreme reaction of Vibrio fischeri to Diazinon (Table 4).
Malathion
Malathion 57% at concentration of 100, 20, 10, 1, 0.01, 0.001 ppm was prepared.
The reaction of Vibrio fischeri light to Malathion provide information
that show an increase trend from 0.001 to 100 ppm, however, this increase is
not sensible, but it shows a great fall in comparison of maximum bioluminescence.
The percentage of light reduction between 0.001 ppm and maximum bioluminescence
is about 97% (Table 5).
| Table 4: | Effect of Diazinon on bioluminescence of Vibrio fischeri |
| SD: Standard deviation, RLU: Relative light unit | |
| Table 5: | Effect of Malathion on bioluminescence of Vibrio fischeri |
| Table 6: | Effect of Selenium on bioluminescence of Vibrio fischeri |
| Table 7: | Effect of Marcury on bioluminescence of Vibrio fischeri |
Selenium
Water spiked with SeO2 to give the concentration of 100, 20,
10, 1, 0.1, 0.001 ppm Se4+. Bacteria light at 0.001 ppm selenium
reduced 95% in comparison of maximum bioluminescence. As an overall look the
light have a decrease trend in different concentration of selenium from 0.001
to 100 ppm, but there is an increase from 0.01 to 0.1 ppm that may show the
mistake in measurements (Table 6). EPA have set the limit
of selenium in drinking water 0.05 ppm.
Mercury
The Environmental Protection Agency (EPA) and Food and Drug Administration
(FDA) have set the limit of mercury in drinking water 2ppb.
Water spiked with HgCl2 to give the concentration of 100, 20, 10, 1, 0.1, 0.001 ppm Hg2+. Bacteria light at 0.001 ppm mercury reduced 96% in comparison of maximum bioluminescence (Table 7).
DISCUSSION
The effect of metal ions and organophosphorus pesticides on luminescence can be investigated. While maximum bioluminescence occurred in 157717 the decrease of light is obvious. In all cases when we move from 100 to 0.001 ppm the bioluminescence is decreased at 0.001 ppm about 95-97% compared with maximum bioluminescence.
The obtained results revealed a dramatic decline in bioluminescence however the amount of biocide is too small and luminometer is very sensitive to measure bioluminescence. The obtained results showed that 2 min is a small time when it compared with Mass Spectroscopy (MS), High Performance Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) (Becvar and Becvar, 2002). Also, our new method is comparable with these old methods because they are complex, time consuming, expensive and require sample pretreatment.
Also the results showed that LOD for this method is about 0.001 ppm when it compares with AAS method in detection of Mercury with LOD 0.01 ppm (Moreno et al., 2009) and HG-AAS method in detection of Selenium has shown LOD 0.04 ppm (Anthemidis, 2006).
In addition it was the first time that the effect of biocides on whole bacteria Vibrio fischeri tested with luminometer, in previous methods luciferase extracted from Vibrio fischeri and then after preparation, the effects of biocides was evaluated. The more future experimental data on other biocides can continue and confirm these data.
The data indicates that it's possible to detect the limit of biocides even more than that EPA set.
CONCLUSIONS
Since the majority of this research depend on optimization of media culture, the media culture was selected which take a short time to prepare in a way to be applicable for a long- term, so the DSMZ MEDIA CULTURE for liquid culture is recommended and for solid culture, Sea Water Agar, is recommended.
As it is mentioned before, optimization of media cultures and the temperature of bacteria growth that can be used for biological element of biosensor for detection biocides in water, so 18°C is the temperature that bacteria grows well and has the most bioluminescence light and we can see the light after 24-48 h.
The experimental results showed that limit of detection (LOD) in Vibrio fischeri for all of the above toxic material is about 1 ppb. Because the traditional methods are not as precise as this method, the present work demonstrated the feasibility of using Vibrio fischeri for detecting biocides.
ACKNOWLEDGMENT
The authors are thankful to Dr Saman Hossein Khani the supervisor at Tarbiat Modares University for using luminometer in biochemistry laboratory.