Analytica Chimica Acta 471 (2002) 113120

Improved detection of toxic chemicals using bioluminescent bacteria

Stefano Girotti a, , Luca Bolelli a , Aldo Roda b , Giovanna Gentilomi c , Monica Musiani c
a

Complex Unit of Chemical, Radiochemical and Metallurgical Science, University of Bologna, Via Belmeloro 8, I 40127 Bologna, Italy b Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 8, I 40126 Bologna c Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Via Massarenti 9, I 40138 Bologna, Italy Received 1 November 2001; received in revised form 7 August 2002; accepted 12 August 2002

Abstract A sensitive, rapid and simple bioluminescent (BL) assay using bioluminescent bacteria to detect the toxic activity of several chemicals is described. This assay is based on the measurement of inhibition of light production of a bioluminescent bacterial strain, isolated from seawater, in the presence of different toxins like heavy metals, organic chemicals, such as benzene, toluene, ethylbenzene, xylene (BTEX) and a wide range of pesticides in environmental samples. The improvement with respect to other commercial and non-commercial bioluminescent assays consists of the possibility to work at room temperature without the need to thermostat, thus allowing the use of simpler and low cost instruments, or to improve the assay using a microplate format, which makes it possible to analyse several samples also continuously for several hours. Using lyophilised bacteria, the assay is performed in less than an hour, without any bacterial cultivation, which makes the test suitable for rapid and sensitive evaluation of chemical pollutants in environmental samples. 2002 Elsevier Science B.V. All rights reserved.
Keywords: Bioluminescent bacteria; Heavy metal; Pesticide; BTEX; Biotoxicity assay

1. Introduction Luminous bacteria are the most abundant and widely distributed of the light-emitting organisms and are found in marine, freshwater, and terrestrial environments [13]. These bacteria are all Gram-negative motile rods and can function as facultative anaerobes. To date fewer than 1% of the known species have been studied in detail and most information concerns the marine bacteria of three genera: Photobacterium,
Corresponding author. Tel.: +39-051-242052/251147; fax: +39-051-249770. E-mail address: girotti@biocfarm.unibo.it (S. Girotti). URL: http://biocfarm.unibo.it/girotti/

Vibrio, and Photorhabdus, mainly Photobacterium (Vibrio) scheri and Vibrio harveyi [4]. Toxic pollutants are found everywhere, in water, in the air and in the soil, causing environmental damage. Early detection of pollutants is therefore increasingly important in order to avoid the use of animals for toxicity testing. For ethical and economic reasons, various techniques have been developed and proposed as potential alternatives, among them the luminescent bacteria toxicity test [5]. Bioluminescent bacteria emit light when they nd themselves in an optimal environment. In vivo luminescence is a sensitive indicator of xenobiotic toxicity to micro-organisms because it is directly coupled to respiration via the electron transport chain, and thus

0003-2670/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 8 7 0 - X

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reects the metabolic status of the cell. If noxious substances are present, the luminescence decreases. The higher the degree of toxicity, the less the amount of light emitted by the bacteria. Thus, the presence of toxic substances can be evaluated and several commercial kits and dedicated instruments have been available for some time. Compared with other bioassays, the luminescent bacteria toxicity test proved that its average sensitivity is within the same order of magnitude as the other tests in evaluating organic or inorganic pollutants [6,7]. It is, however, acknowledged that the battery of test approach, using several different short-term biological tests, is preferred in any monitoring scheme. The European Standard EN ISO [8] describes the methodology for determining the inhibition of bioluminescent light emission from sea bacteria species Vibrio scheri (NRRLB-11177); following these procedures the analysis must be performed using reagents and measurement chamber at a strictly controlled temperature (15 1 C), thus using dedicated instruments. This methodology is applicable for monitoring wastewater, deep and supercial fresh water, salt water and water extracts. In order to facilitate in-eld measurements, we developed an assay which could be performed using simple and low cost instrumentation without thermostatic controls (at room temperature), giving results in a few minutes. In our work, the culture, storage and the reconstitution of a Vibrio logei strain, isolated from the waters of the Mediterranean Sea, were optimised to have standardised reagents to perform the assays. All the tests were done using a luminometer with a 96-well microplate which could be used to run multiple assays at a time. The presence and concentration of many metals, such as lead, mercury and zinc, toxic organic chemicals like benzene, toluene, ethylbenzene and xylene (BTEX) and a wide range of pesticides in the aqueous system were analysed according to the threshold values established by European and Italian legislation. The assay was also applied in environmental, river and seawater samples. 2. Experimental 2.1. Growth, maintenance and preservation of bacterial strain A luminescent bacterial strain (Vibrio logei) isolated from seawater was used for all experiments. Bacterial

cultures were grown for 18 h at 20 1 C in ALNa broth (AB) containing 5 g/l of bacto peptone, 3 g/l of bacto-yeast extract, 30 g/l of NaCl, 104 M CaCl2 , 105 M MgCl2 , pH 7.07.2 or in LuriaBertani (LB) broth [9]. In brief, luminescent bacteria were cultured under sterile conditions in Petri dishes, containing ALNa agar (AA, i.e. ALNa broth added with 12 g/l of bacto-agar) for stock cultures. The dishes were incubated for 18 h at 20 1 C, and the luminescent single colonies were then selected by means of visual observation in the dark. An aliquot of 10 ml of AB was inoculated with one single luminescent colony and after shaking at 180 rpm for 18 h at 20 1 C, the optical density of the culture was determined by spectrophotometric assay at 550 nm and a stock suspension (0.25 absorbance units equivalent to 3 108 cells/ml) was prepared. Preservation of the bacterial strain was performed by freezing treatment at 80 C or by lyophilising treatment. As far as freezing is concerned, aliquots of 100 l of bacterial stock suspension were prepared in protective medium adding 15% of glycerol to AB and immediately frozen at 80 C. For the lyophilising process, more concentrated suspensions were used; the higher concentration of bacteria was optimised to balance the luminescence decrease after the lyophilisation process and resulted in 12 108 cells/ml suspended in a lyoprotecting medium (12% w/v lactose, 2% w/v soluble starch, 1% w/v NaCl, pH 7.5). In brief, luminescent bacteria grown in AB for 18 h were centrifuged at 5000 rpm for 5 min and the pellet was resuspended in cold (4 C) 3% NaCl solution. This suspension was recentrifuged under the same conditions and the pellet was resuspended at the same concentration of 12 108 cells/ml in cold (4 C) lyoprotecting medium. After 5 min at 4 C, the suspension was shaken or vortexed to resuspend all the bacteria, aliquoted in vials containing 100 l each and stored at 20 C for almost 1 h. These frozen aliquots were ready to be lyophilised at 1 103 mbar for 24 h at a low temperature (approximately 30 C). The lyophilised bacteria were stored at 20 C and in these conditions were active for almost 3 months. To perform the bioluminescence assay each aliquot of the freeze-dried bacteria was reconstituted with 100 l per tube of distilled water; if many lyophilised bacteria tubes were reconstituted for a

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single assay all the suspensions were collected and mixed. Yeast extract, bacteriological agar and peptone were from Biokar Diagnostics. Lactose, soluble starch, MgCl2 , CaCl2 , glycerol, and NaCl were from Merck. 3,5-Dichlorophenol was from Aldrich. 2.2. Instrumentation of bioluminescence assay Luminescent measurements were performed using a Wallac Victor 1420 Multilabel Counter luminometer (Wallac, Sweden). Before starting the assays, light emission was measured on the BL bacteria in microplate wells to determine if the light level was optimal for the analysis, at least 200300 relative light units (RLU) with respect to 20 RLU for a well without bacteria. The analyses were performed by continuously scanning the light emission of the plate and stopping when a signicant difference between the blank and the analyte signals appeared; this can vary from analyte to analyte, usually 3060 min. This also makes it possible to follow light inhibition for several hours, mainly overnight. 2.3. Bioluminescence assay for heavy metals Standard solutions of chlorides of heavy metals (from Merck) were prepared in 3% NaCl, or in 1.5% NaCl + 10% glucose. Pb, Cd and Hg were in a concentration range from 0.0025 to 17 ppm and Cu, Cr, Zn from 0.01 to 10 ppm. These chemicals were from Carlo Erba. An amount of 180 l of each metal sample or blank solution (3% NaCl) were added to 20 l of reconstituted lyophilised bacteria suspension placed in a well of a microtiter plate, and the bacterial luminescence was measured and recorded for 30 min in 5 min steps. Results were expressed as inhibition percentage with respect to the blank emission. The values of analyte concentration able to inhibit the bacterial light emission by almost 50 and 20%, considering the blank signal as 100%, were expressed as EC50 and EC20 , respectively. 2.4. Bioluminescence assay for BTEX Stock solutions of BTEX (0.000860 ppm in methanol), from Merck, were used to prepare work-

ing solutions at different percentages of methanol in aqueous 3% NaCl of the BTEX. The analysis was performed using the same volumes and procedures as for heavy metals, the blank was methanol at different percentages in 3% NaCl and the bacterial luminescence was measured and recorded for an hour in 5 min steps. 2.5. Bioluminescence assay for pesticides Several pesticides, using the same conditions as for the heavy metal assay, were tested: Chlorpyrifos, Molinate, Carbaryl, Chlordan, Mercaptodimethur, Endosulfan, p,p -DDT [1,1,1-trichlor-2,2-bis-(4-chlorophenyl)-ethan)], o,p -DDT [2-(2-chlorophenyl)-2(4-chlorophenyl)-1,1-trichloroethan], p,p -DDE [2, 2-bis-(4-chlorophenyl)-1,1-dichloroethan], p,p -DD D [2,2-bis-(4-chlorophenyl)-1,1-dichloroethan], o,p DDD [2-(2-chlorophenyl)-2-(4-chlorophenyl)-1,1dichloroethan], o,p -DDE [2-(2-chlorophenyl)-2-(4chlorophenyl)-1,1-dichloroeten], Parathioethyl, Endrin, Endrin aldehyde, Aldrin, Dieldrin, alpha-Endosulfan, beta-Endosulfan and Heptachlorepoxide. All pesticides were from Riedel de Hen. Stock pesticide solutions (1 mg/ml methanol) were diluted with aqueous NaCl to obtain different concentrations of pesticides, all in a nal percentage of methanol of 3% in 3% NaCl, and the blank was 3% methanol in 3% NaCl. Different measuring times were adopted (up to 60 min) to highlight differences between the blank and the samples. 2.6. Bioluminescence assay for river and seawater River water toxicity was analysed, as for the heavy metal assay, using non-muddy water samples coming from industrial and non-industrial zones. Due to the low osmolarity of the samples these were supplemented with NaCl to obtain a nal concentration of 3% and avoid unwanted bacterial lysis; the blank was 3% NaCl. The bacterial luminescence was measured and recorded for 30 min in 5 min steps. 3. Results and discussion 3.1. Optimisation of bacterial cultivation As a rst test for the present study, different conditions for bacterial growth, such as culture temperature

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Fig. 1. (- - -) Measurements of luminescence (RLU) and () absorbance (ABS) during a bacterial culture at 20 C in AB medium.

(20 and 37 C), culture times (1248 h), media (LB, AB and seawater) and culture exposure to the light, were analysed. The optimal conditions of 20 C of culture temperature, 18 h of culture time, the use of AB medium and the culture exposure to the light were achieved. Fig. 1 shows the results of a luminescent bacteria culture at 20 C in AB medium to determine the course of luminescence and absorbance. The absorbance value is correlated to the bacteria number and indicates bacterial growth, the luminescence is correlated to the bacteria number and to luminescent efciency. As shown in Fig. 1, the luminescence peak corresponds to bacterial growth stabilisation and in the logarithmic growth the luminescence starts to increase; therefore in order to obtain the highest luminescence it is important to use the bacteria after 1617 h of incubation at 20 C. Different preservation conditions of bacterial strains, such as freezing or lyophilising treatments, were also evaluated. For the lyophilising treatment, two variables were investigated: the pH of the medium and the contact time between medium and bacteria. The best results were obtained when the contact time was 5 min at pH 7.5. For the freezing treatment, the best results were obtained by the freezing treatment at 80 C. In these conditions, bacterial suspensions were stable for 612 months, without loss of viability or luminescence properties when thawed or reconstituted. Table 1 reports the comparison of the EC50 obtained with our

bacteria of standards (3,5-dichlorophenol, zinc sulfate and potassium dichromate) used in the ISO [8] procedure for freeze-dried bacteria, and the agreement is good. The optimised growth and preservation conditions of bacteria were used for the assay of heavy metals, BTEX, pesticides, river waters and seawaters. 3.2. Microplate assay In addition to determining the EC50 and the EC20 of the various analytes, the microplate assay makes it possible to follow the trend and type of inhibition that each toxic compound has on BL bacteria. This is an important aspect that cuvette assays do not allow. In order to use this system, it is fundamental not to have to use thermostatic controls, thus using simpler instruments. This is, however, not possible
Table 1 Comparison of the EC50 obtained with our bacteria of standards (dichlorophenol, zinc sulfate and potassium dichromate) used in the EN ISO 111348-3:1999 procedure for freeze-dried bacteria Analyte EC50 (ppm) ISO 3,5-Dichlorophenol Cr6+ Zn2+ 3.36 0.32 18.71 6.17 2.17 0.73 Found 3.5 0.4 5.0 0.5 3.0 0.1

The values are the average of three measurements S.D.

S. Girotti et al. / Analytica Chimica Acta 471 (2002) 113120 Table 2 Limit of detection (LOD) and EC50 of heavy metals by BL assay and limits for Italian legislation [10] Heavy metal Pb(II) Hg(II) Cd(II) Zn(II) Cr(VI) Cu(II) LOD (ppm) 0.1 0.005 0.03 1 0.2 0.2 EC50 (ppm) 0.2 0.05 0.5 3.0 5.0 0.3 Limits for surface water (ppm) <0.2 <0.005 <0.02 <0.5 <2 <0.1 Limits for wastewater (ppm) <0.3 <0.005 <0.02 <1 <4 <0.4

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with the majority of commercially available bacteria. In fact, inhibition experiments with these bacteria present very high (80130%) reproducibility values when used with the microplate system, while the strain that we used presents lower values, as also shown in Table 1 for standard 3,5-dichlorophenol, zinc sulfate and potassium dichromate solutions. 3.3. Heavy metal analysis In the analysis of heavy metals, the limit of detection (LOD) of the tested metals, at the 3 level, revealed by a signicant decrease in bacterial luminescence at room temperature after 10 min of contact, was below or similar to the threshold values established by Italian laws on water analysis (Table 2) [10]. Imprecision of the results in Table 2, expressed as coefcient of variation, was in the range 819%. As an example of

the effect of heavy metals on luminescent bacteria, Fig. 2 reports an assay on mercury standard solutions at different concentrations using lyophilised bacteria. A signicant decrease in bacterial bioluminescence was found at a concentration of 0.00510 ppm, detectable after 5 min contact with the metal. The use of lyophilised bacteria gave a constant blank signal which is useful for signal comparison. Other experiments were performed using frozen bacteria and similar results were obtained, but a higher imprecision (76133%, expressed as coefcient of variation) was obtained with respect to that of lyophilised bacteria (1040%). For this reason, lyophilised bacteria were used in subsequent experiments. Some authors [11] suggest the addition of glucose to the suspension to increase the bacterial sensitivity to heavy metals; different solutions of glucose in NaCl were tested and with these conditions good results were obtained with NaCl 1.5%glucose 10% (LOD of 1 ppm for lead and mercury). NaCl without glucose detected lower concentrations of metals (LOD of 0.1 ppm for lead and 0.005 ppm for mercury) and 3% NaCl was therefore used to adjust the salinity of the samples in all subsequent experiments. Having shown the different detection limits of the single metals, experiments mixing metals at concentrations below each detection limit and diluting to reach a total concentration always below the limit concentration of a single metal were performed. In these conditions, a synergic effect of the metals on bacterial

Fig. 2. BL assay on mercury standard solutions at different concentrations using lyophilised bacteria. Bars represent standard deviation (n = 3). ( ) Blank, ( ) 0.005 ppm, (, ) 0.1 ppm, ( ) 0.5 ppm, ( , ) 1 ppm, ( ) 5 ppm.

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Fig. 3. BL assay of different dilutions of metal mixture: mercury 0.001 ppm, chrome 0.53 ppm, iron 0.44 ppm, zinc 0.47 ppm, lead 0.07 ppm, aluminium 0.20 ppm, copper 0.07 ppm lower than legal limit concentrations. Bars represent standard deviation (n = 3). ( ) Blank, ( ) 1:50, ( ) 1:10, (, ) 1:5, ( ) 1:2.

luminescence was found as reported in Fig. 3 for the dilution 1:50. 3.4. BTEX analysis The bioluminescent assay using lyophilised bacteria in 3% NaCl at room temperature was then used to detect BTEX concentrations. BTEX compounds were solubilised in methanol/3% NaCl solutions to avoid the normal methanol toxicity on bacteria. The optimal concentration of methanol for 1 h assays of BTEX was 3%; more concentrated methanol solutions were too toxic for the bacteria and the blank signal after 60 min was at the background level. The results of bacterial luminescence assay performed for 30 min on BTEX showed that the inhibition scale was: toluene = xylene > ethylbenzene > benzene. The EC50 limits were: toluene = 0.2 ppm; xylene = 0.2 ppm; ethylbenzene = 0.3 ppm; benzene = 15 ppm. The limits of detection for ethylbenzene and benzene were higher than the legal limits xed for wastewater in Italy (0.2 ppm for river water and 0.4 ppm for public wastewater) [10]. The bacterial sensitivity for toluene and xylene is appropriate to reveal whether these solvents are in the legal range of concentration. If the EC20 value is considered instead of EC50 , only benzene is not detectable by luminescent bacteria in a legal concentration range, so it is not nec-

essary to introduce a concentration/extraction step for the analysis of toluene, xylene and ethylbenzene [12]. 3.5. Pesticide analysis The bioluminescent assay performed on 21 pesticides solubilised in 3% methanol solutions showed that after 30 min of contact at room temperature, only 10 of them signicantly inhibited the bacterial luminescence at a concentration below 400 ppm, as reported in Table 3, together with their EC50 values. Imprecision of the results in Table 3, expressed as coefcient of variation, was in the range 1022%. All these limit concentrations were higher than their water solubility [13], so only after an extraction from
Table 3 Limit of detection (LOD) and EC50 of the analysed pesticides Pesticide o,p -DDD Carbaryl Molinate Mercaptodimethur Dieldrin Chlorpyriphos p,p -DDE p,p -DDD o,p -DDE p,p -DDT LOD (ppm) 20 20 40 40 200 200 200 200 200 200 EC50 (ppm) 40 30 100 100 250 300 250 250 300 250

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Fig. 4. BL assay of river water sample using lyophilised bacteria. Blank (), industrial wastewater ( ). Bars represent standard deviation (n = 3).

Fig. 5. BL analysis of seawater samples. Blank (), industrial wastewater (----). Bars represent standard deviation (n = 3).

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waters should it be possible to reveal pesticides with the bioluminescent method. Considering that an assay takes 4050 min to be performed without a sample treatment like extraction or a similar process, a concentration step will cause a major increase in the procedure time. The bioluminescence assay for pesticides is not therefore suitable for a rapid detection method. Analyses were also performed on mixtures of pesticides but no signicant results were detected. 3.6. Water sample analysis Once the efcacy of the BL assay had been determined on standard solutions of several analytes, river water and seawater samples were analysed for 2 h at room temperature. Fig. 4 demonstrates that efuents of industrial wastewater inhibit bacterial luminescence; this is evident only at more than 60 min of assay when the sample signal decreases with respect to the blank. The river water samples which were not efuents of industrial wastewater, on the other hand, increased the bacterial luminescence (compared to the blank); this is probably due to the presence of nutritive substances which are absent in the blank (3% NaCl). For this reason, it is important to have a representative blank river water sample similar to the one to be analysed. As shown in Fig. 5, the analysis of seawater samples collected near the mouth of a river with industrial estate efuent did not show any bacterial luminescence inhibition; the dilution of the river water with the seawater close to the mouth clearly decreases the toxicity of the samples. Moreover, this assay format, using microplates at room temperature, continuously following light emission for a long time in a simple way, allows the determination of the minimum time inhibition of the sample. 4. Conclusion In conclusion, the bioluminescence assay performed on microplates demonstrated that it is possible to apply biotoxicity assays of heavy metals and BTEX to water samples at room temperature, increasing the rapidity of the response and the easiness of the procedures. The use of a microplate luminometer at room temperature automated the assay without stringent temperature control, allowing several samples to be tested in the same run and making it possible easily follow the

inhibition of the light emission. It is possible to obtain data similar to those of standard solutions according to the ISO procedure [8], including EC50 values, without stringent temperature conditions; the conditions required for the control of lyophilised bacteria are in any case respected, with inhibition in a range of 2080% for standard solutions of 3,5-dichlorophenol [8]. Analysis of eld samples of river water demonstrated that a simple sample treatment without any concentration/extraction step is needed. Further studies will be performed to establish standardised controls for all real samples to be analysed. Acknowledgements This work was supported by grants from the CNR (Consiglio Nazionale delle Ricerche) Biotechnology Project, MURST (Ministero della Universit e della Ricerca Scientica e Tecnologica) and from the University of Bologna (Ricerca Fondamentale Orientata and Funds for Selected Research Topics). Thanks to the Centro Interdipartimentale di Ricerche Biotecnologiche for the use of the luminometer. We thank Dr. M. Anwar Panezai for collection of some data. References
[1] J.W. Hastings, J. Mol. Evol. 19 (1983) 309. [2] D.J. OKane, D.C. Prasher, Mol. Microbiol. 6 (1992) 443. [3] P.V. Dunlap, U. Mueller, T.A. Lisa, K.S. Lundberg, J. Gen. Microbiol. 138 (1992) 115. [4] E.A. Meighen, Annu. Rev. Genet. 28 (1994) 117. [5] G.S.A.B. Stewart, Lett. Appl. Microbiol. 10 (1990) 1. [6] K.K. Kwan, B.J. Dutka, S.S. Rao, D. Liu, . Environ. Pollut. 65 (1990) 323. [7] L.I. Sweet, D.E. Travers, P.G. Mieier, Environ. Toxicol. Chem. 16 (1997) 2187. [8] British StandardBS EN ISO 11348-3:1999, Water quality determination of the inhibitory effect of water samples on light emission of Vibrio scheri (Luminescent bacteria test), part 3, BSI, Index House, Ascot, SL5 7EU, UK. [9] J. Sambrook, D. Russel, Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. [10] Decreto Legislativo 11 maggio 1999, n. 152, Gazzetta Ufciale 246, 20 Ottobre 2000Supplemento Ordinario 172. [11] A.C. Ekvall, G.M. Morrison, Environ. Toxicol. Chem. 14 (1995) 17. [12] A. Fernndez, C. Tejedor, F. Cabrera, A. Chordi, Water Res. 29 (1995) 1281. [13] C. Tomlin, The Pesticide Manual, Incorporating The Agrochemical Handbook, 10th ed., Crop Protection Publication, Surrey, UK, 1994.