Biosensors and Bioelectronics 22 (2007) 13961402

Fibre-optic bacterial biosensors and their application for the analysis of bioavailable Hg and As in soils and sediments from Aznalcollar mining area in Spain
Angela Ivask a,b, , Tal Green c , Boris Polyak c , Amit Mor c , Anne Kahru c , Marko Virta d , Robert Marks c,e
a

National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia b Tallinn University of Technology, Ehitajate tee 5, Tallinn19086, Estonia c Department for Biotechnology Engineering, P.O. Box 653, Beer-Sheva 84105, Israel d Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland e National Institute for Biotechnology in the Negev, P.O. Box 653, Beer-Sheva 84105, Israel Received 30 March 2006; received in revised form 6 June 2006; accepted 15 June 2006 Available online 4 August 2006

Abstract Fibre-optic biosensors for Hg and As were developed by attaching alginate-immobilised recombinant luminescent Hg- and As-sensor bacteria onto optical bres. The optimised biosensors (consisting of seven layers of bre-attached bacteria pre-grown till mid-logarithmic growth phase) enabled quantication of environmentally relevant concentrations of the target analytes: 2.6 g l1 of Hg(II) and 141 g l1 of As(V) or 18 g l1 of As(III). The highest viability and sensitivity for target analyte was obtained when bre tips were stored in CaCl2 solution at 80 C. Applicability of the bre-optic biosensors in parallel to the respective non-immobilised sensors was assessed on 10 natural soil and sediment samples from Aznalcollar mining area (Spain). On the average 0.2% of the total Hg and 0.87% of the total As proved bioavailable to bre-attached bacteria. Interestingly, about 20-fold more Hg and 4-fold more As was available to non-immobilised sensor bacteria indicating the importance of direct cell contact (possible only for non-immobilised cells) for enhanced bioavailability of these metals in solid samples. 2006 Elsevier B.V. All rights reserved.
Keywords: Mercury; Arsenic; Pollution; Recombinant bioreporter bacteria; Bioluminescence; Fibre-optic biosensor

1. Introduction Mobilisation of heavy metals from the Earths crust by continuously increasing industrial activities has caused environmental problems worldwide. Due to the sorption of metals on solid matrix of soils and sediments these environments are major sinks for the released metals. The traditional approach for evaluating heavy metal pollution in soils and sediments is based on physico-chemical analysis that allows accurate and sensitive quantication of metals. However, this analysis is complex, costly and requires specialized laboratories. In addition, such methodologies fail to provide data on the bioavailability of metals in solid environments/samples and on their behaviour in

Corresponding author. Tel.: +372 6398382; fax: +372 6398382. E-mail address: angela@kb.ee (A. Ivask).

mixtures (Belkin, 2003). The latter issues could be overcome by the use of living test-organisms. A number of non-specic biotests, measuring the general toxicity of the sample, have been developed for evaluation of the hazard of bioavailable environmental pollutants. For the specic determination of bioavailable metals, recombinant bacterial sensors offering a possibility for fast and cost-effective analysis of various (e.g., solid phase) samples have been developed. Such sensors report on the presence of a metal by increasing the signal (e.g., luminescence) that is controlled by a protein specically recognizing this metal (K hler o et al., 2000). Most of the work on metal-specic bacterial sensors has been done by using a liquid suspension of viable sensor bacteria. However, a more advanced approach is to use these bacteria in the biosensor system, e.g., by immobilising the cells onto optical bres connected to a photo detection device. This type of bre-optic biosensors have been previously constructed for Cu

0956-5663/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2006.06.019

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(Corbisier et al., 1999), genotoxicants (Polyak et al., 2000), or general toxicity of industrial efuents (Horsburgh et al., 2002). These bre-optic sensors can easily be brought to the eld and used for on-line monitoring. Despite of the in-built advantages of bre-optic biosensors for eld testing (Hale et al., 1996; Marks et al., 1997), their use in on-site monitoring has so far been mainly restricted to aqueous samples. However, according to our experience, the analysis of solid samples like soils or sediments, with bre-optic biosensors is a feasible approach. The aim of this study was to develop bre-optic biosensors for the analysis of environmental samples, e.g., soils and sediments in situ. For that, the existing recombinant luminescent Escherichia coli MC1061 (pmerRluxCDABE) and MC1061 (parsluxCDABE) (Hakkila et al., 2002) responding specically to Hg and As, respectively, were immobilised onto optical bres in order to develop self contained biosensors. The system was optimised for the Hg biosensor and the derived protocol was used for analysing bioavailable Hg and As in natural soil or sediment suspensions. The samples originated from the Aznalcollar mining area (Spain) and were analyzed during SENSPOL Technical Meeting on Sensors for Characterisation and Monitoring of a Contaminated Site (69 November 2002, Sevilla, Spain). The bioavailable amounts Hg and As were quantied and compared with the results obtained with non-immobilised sensor bacteria. Optimal storage conditions of bre-optic sensors were seeked for as longer shelf-life (of the bre-optic biosensors) would remarkably simplify their future use for eld testing.

inducible by heavy metals (Leedj rv et al., 2006) was used for a taking into account colour/turbidity or any putative adverse or stimulating effects of the samples on the luminescence of bacteria. No control bacteria were used for the bre-optic metal biosensors, as the interference of sample colour or turbidity does not affect the collection of luminescence by optical bres. 2.2. Attachment of sensor bacteria onto optical bres Multimode optical bres, SFS400/440 (Fibreguide Industries, Inc., USA) were used for immobilisation of the sensor bacteria. Prior to preparation, black nylon jackets covering the bres were stripped away from a 1-cm long optical bre tip and the bre tip was thinned to 350 5 m (Polyak et al., 2000) by incubation in 48% HF for 10 min. The thinned bre tips were washed with 70% of ethanol before the attachment of bacteria. Sensor bacteria, that were to be immobilised, were continuously shaken overnight at 37 C in M9 minimal medium (Sambrook et al., 1989) with glucose and 0.5% of hydrolyzed casein (Hg sensor E. coli MC1061 (pmerluxCDABE)) or LuriaBertani medium (Sambrook et al., 1989) (As sensor MC1061 (parsluxCDABE)) containing 100 g ml1 of ampicillin. One hundred-fold diluted overnight culture was grown until certain cell density (20110 Klett units, whereas 40 corresponding to OD600 of 0.35 was found to be optimal). The bacteria were then mixed 1:1 with lter-sterilized 2% (w/v) low viscosity sodium alginate (Sigma, A-2158) aqueous solution. The 1-cm optical bre tip was dipped (for a few seconds) to the bacterialalginate suspension and then (for a few seconds) into sterile 0.5 M CaCl2 solution. The procedure resulted in the formation of solid Caalginate matrix that attached the alginateentrapped bacteria onto the bre. The procedure was repeated several times in order to increase the bacterial covering. After preparation, the bres were incubated in CaCl2 solution for 25 min. Performance of 611 layers of immobilised bacteria was tested, however the covering of seven bacterial layers was used in most of the experiments. For Hg diffusion studies, HgCl2 was attached onto the bres together with bacteria (as bacteriaalginateHgCl2 mixture). The bre-optic sensors were either used immediately after preparation (in most of the experiments) or stored under different conditions (see below). The number of viable bacteria on bres was estimated by dissolving the solid Caalginate matrix with 1 ml of 50 mM sodium citrate and cultivating the detached

2. Materials and methods 2.1. Recombinant sensor bacteria The used recombinant sensors comprised of the host E. coli MC1061 carrying the pmerRluxCDABE plasmid (Hg-sensor) or parsluxCDABE plasmid (As-sensor) (Table 1). These plasmids have been constructed by Hakkila et al. (2002) and contain fused sensing (metal-recognizing protein) and reporter elements (luxCDABE operon from Photorhabdus luminescens). The Hg and As sensors are highly specic towards the target analyte: no other metal has been shown to induce the luminescence in As sensor (Tauriainen et al., 1999) and only Cd has been shown to co-induce the luminescence in Hg sensor, but at 50 times higher concentrations compared to Hg (Virta et al., 1995). The luminescent recombinant E. coli MC1061 (pDNlux), that does not contain the metal-sensing elements and is therefore, non-

Table 1 Description of the sensors used in this study. Host bacterium for all sensors was Escherichia coli MC1061 Sensor Hg As Non-inducible control bacterium Sensor/control plasmid (reference) pmerRluxCDABE (Hakkila et al., 2002) parsluxCDABE (Hakkila et al., 2002) pDNlux (Leedj rv et al., 2006) a Format of the assay Sensor bacteria immobilised on optical bres Non-immobilised (free) sensor bacteria Sensor bacteria immobilised on optical bres Non-immobilised (free) sensor bacteria Non-immobilised (free) bacteria Limit of quantication ( g l1 ) (R.S.D.) 2.6 0.9 0.03 0.001 141 32 [As(V)], 18 4.1 [As(III)] 80 1.6 [As(V)], 8 0.2 [As(III)] Not responding to heavy metals

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bacteria on LB (1.5% of agar (w/v)) plates supplemented with 100 g/ml of ampicillin overnight at 37 C. 2.3. Effect of storage conditions on performance of the immobilised sensor bacteria The effect of storage medium and temperature on performance (inducibility with target analyte) and viability of immobilised sensor bacteria during 73-day of storage was studied. Altogether three different storage media were studied: 0.15 M CaCl2 , LB medium (Sambrook et al., 1989) (NaCl in LB medium was replaced by equimolar concentration of CaCl2 to ensure the attachment of the hydrogel probe onto the optic bre tip) or M9 medium (Sambrook et al., 1989) supplemented with 0.5% of hydrolyzed casein. The following storage temperatures were applied: 80, 20 C (30% (v/v) of glycerol was added as cryoptotectant), +4 C and ambient (22 C, only for CaCl2 ). For viability and performance studies of the deep-freeze stored biosensors the medium surrounding the bres was thawed and replaced by 5 ml of the respective fresh medium followed by recuperation for 15 min at room temperature prior to measurement. 2.4. Non-immobilised bacteria Non-immobilised (free) sensor and the respective noninducible control bacterial strains were pre-grown to OD600 of 0.6 in conditions as described above, lyophilised and rehydrated prior to testing as described by Ivask et al. (2002). 2.5. Preparation of soil and sediment samples Pre-treatment of the samples (air-drying and homogenisation to a particle size around 100 m) and determination of the total heavy metal content by ICP-OES and ICP-MS after digestion with aqua regia was done by the organisers of the Technical Meeting on Sensors for Characterisation and Monitoring of a Contaminated Sites (69 November 2002, Sevilla, Spain). Before bioanalysis all the samples were mixed with water in a ratio soil/sediment:water (w/v) of 1:5 (for the analysis with breoptic biosensors) or 1:9 (for the analysis with non-immobilised sensor bacteria). The obtained suspensions were vortexed for 1 min and geometrically diluted (dilution factor 3) with water up to 1000 times. 2.6. Test protocol Luminescent response of the bre-optic biosensors was measured after incubation at room temperature for 3 h (in optimisation studies also 0.54 h were tested) in either 500 l of environmental sample, water (control) or standard solution of HgCl2 , NaAsO2 or Na2 HAsO4 7H2 O (all Sigma). This incubation is necessary for target metal-induced synthesis of luciferase by sensor bacteria. When sensor bacteria were immobilised onto bre tips together with HgCl2 (Hg diffusion study) the incubation time started from the moment the bacteria were mixed with the HgCl2 alginate mixture prior to immobilisation. The lumi-

nescence was measured by photon counting system and analyzed by special software as described by Polyak et al. (2000). Two to three independent measurements were performed for every sample or standard. The detailed measurement procedure for non-immobilised bacteria was previously described by Ivask et al. (2002). Briey, 100 l of the reconstituted bacterial suspension (24 106 viable cells) was mixed with 100 l of water, metal dilution or environmental sample in multiwell plates and incubated at room temperature for 2 h. At least three independent measurements were performed with every sample/standard. The luminescence was measured on 425-014 Triathler Luminometer (Hidex Oy, Turku, Finland). The response of the immobilised and non-immobilised sensor bacteria was calculated as follows: induction = LS LB (1)

where LS is the luminescence of the sensor bacteria incubated in a sample and LB is the luminescence of the sensor bacteria incubated in water (background luminescence). Induction of non-immobilised bacteria in a sample was multiplied by the correction factor (CF), obtained from measurements with control bacteria and calculated as follows: CF = LB LS (2)

where LS and LB are the luminescence of control bacteria in the sample and water, respectively. The limits of quantication of the sensors for the target analyte (Table 1) were calculated according to the induction of the sensor bacteria by standard solutions (HgCl2 , NaAsO2 and Na2 HAsO4 7H2 O) and variation of their background luminescence (error of the assay) by reading the concentration of the respective metal that corresponded to the lowest statistically signicant induction of the calibration curve (Hakkila et al., 2004). Calibration of the sensors with the target metals was performed together with each run of environmental samples. The fraction of bioavailable metals was calculated by linear regression of the calibration curve and the induction of luminescence by environmental sample. 3. Results and discussion 3.1. Optimisation of the optical bre biosensor system All the optimisation studies for the bre-optic biosensor system were done by using Hg-sensor bacteria E. coli MC1061 (pmerRluxCDABE) as a model. 3.1.1. Physiological conditions (growth phase) of test bacteria In order to nd the optimal physiological conditions of the sensor bacteria before their immobilisation onto optical bres the bacteria were cultivated to different cell densities (corresponding to different growth phases), immobilised and their luminescent response to HgCl2 standard solution (0.13 mg l1

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Fig. 1. (A) Photo of immobilised bacterial adlayers on optical bre tip; (B) enlarged image of the immobilised adlayers: the thicknesses of the polymerised alginatebacterial layers are between 120150 m; (C) luminescent response of the Hg-biosensors composed of different number of bacterial adlayers: 6 ( ), 7 ( ), 8 ( ), 9 ( ), 10 (), 11 ( ) or 12 ( ), to HgCl2 standard solution (0.13 mg l1 of Hg2+ ) after induction from 15 min to 4 h. Data represent mean S.D. for three measurements.

of Hg2+ ) was measured. The highest response (up to 37-fold induction of luminescence after 3.5-h incubation with HgCl2 , data not shown) was obtained when the bacteria were in early logarithmic growth phase (OD600 = 0.35). This cell density was used in all the following experiments. 3.1.2. Number of bacterial adlayers on bres The optimal number of bacterial adlayers on optical bres was investigated by covering the bre tips with 611 layers of alginate immobilised sensor bacteria (see Section 2) and exposing the resulting bre-optic biosensors to 0.13 mg l1 of Hg2+ . The thickness of each layer, determined by microscope, was about 130150 m (Fig. 1B). Fig. 1C shows mercury-induced luminescence of bre-optic biosensors consisting of different number (611) of layers of alginate-entrapped sensor bacteria. The luminescence increased in parallel to the incubation time and, as a rule, decreased with the increasing number of bacterial layers. Fibres with seven layers proved the most sensitive and were used in further experiments. The poorer response of bres covered with thicker cell layer could be explained by the hindered contact between the inducing metal and the sensor bacteria in inner layers, contributing most to the light output of the

biosensor. Thus, the effect of alginate matrix on mobility (diffusion) of Hg2+ was studied by comparing the time needed for the induction of the luminescence of bre-optic sensors immobilised with or without HgCl2 . As expected, the sensor tips, where Hg was co-immobilised in alginate matrix showed faster luminescent response to mercury. The approximate time needed for the diffusion of Hg into alginate matrix in concentration causing two-fold induction of the mercury biosensor was 90 and 40 min for the tested 0.002 and 0.007 mg l1 of HgCl2 (data not shown). 3.1.3. Exposure time In order to induce the bioluminescence of biosensors the metal ions have to diffuse into the alginate matrix and enter bacterial cells. For analysing the kinetics of these processes and for determining of the optimal incubation time the immobilised Hg sensor bacteria were exposed to Hg concentrations of 0.0020.7 mg l1 during up to 5 h. As seen in Fig. 2A in case of all the tested Hg concentrations, the luminescence of the biosensor increased during 4 h and remained stable or even decreased after that. Thus, 4 h was obviously enough for equilibrium between Hg ions in alginate and the surrounding solution as

Fig. 2. (A) Kinetics of induction of the luminescent response of bre-optic Hg biosensor. Sensors were incubated with different concentrations of Hg2+ (mg l1 ): 0.002 (), 0.007 (), 0.02 ( ), 0.07 ( ), 0.2 ( ), and 0.7 ( ). Vertical dotted line indicates the 3-h incubation time considered optimal for further measurements; (B) concentrationresponse curve for bre-optic Hg biosensor ( 3-h incubation time, data plotted from panel A) ( ) and non-immobilised (free) sensor bacteria (incubation time 2 h) ( ). Data represent average S.D. for three independent measurements performed in triplicates (in the case of bre-optic sensors) or duplicates (in the case of non-immobilised free sensor bacteria).

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well as between the intracellular Hg and Hg ions in the alginate matrix. In order to obtain maximum response of the biosensor, while remaining in the region where the luminescence signal is still increasing, 3 h was considered optimal and was used in further experiments. 3.2. Concentrationresponse curve of the bre-optic Hg biosensor Considering all the parameters that were chosen for optimising the performance of the bre-optic sensors (growth phase of sensor bacteria, the number of alginate-bacteria layers on bres and exposure time of bre-optic sensors with sample) the following conditions were chosen: OD600 = 0.35 of sensor bacteria before immobilisation, seven immobilised layers on optical bres and 3-h exposure time in the assay. The performance of these optimised bre-optic sensors was evaluated with 0.00026 mg l1 of Hg2+ (Fig. 2B). The bioluminescent response started at 0.001 mg l1 of Hg2+ , reached maximum (about 200-fold induction of luminescence compared with the value obtained for water) at 0.03 mg l1 of Hg2+ and decreased starting from 0.07 mg l1 of Hg2+ , obviously due to the toxicity. It should be mentioned that Hg is one of the most toxic heavy metals due to its high afnity for sulphydryl (SH) groups in proteins. Indeed, 0.1 and 0.2 mg l1 of Hg decreased the number of viable sensor bacteria on bres approximately by 50% after 4.5 h of incubation and 1 mg l1 of Hg had killed all the bacteria by that time (data not shown). Fig. 2B shows that the linear detection range of the Hg biosensor was between 0.002 and 0.02 mg l1 of Hg2+ . The calculated limit of quantication of the immobilised sensor for Hg2+ was 2.6 g l1 (see also Table 1). In parallel, HgCl2 standards were also used to induce the non-immobilised (free) E. coli MC1061 (pmerRluxCDABE) (Fig. 2B). The non-immobilised bacteria were about 100 times more sensitive towards mercury than the bre-attached bacteria (Fig. 2B) showing that only about 1% of Hg entering the sensor cells in aqueous solution (in case of non-immobilised bacteria) is accessible to alginate-entrapped bacteria. This may be due to greater permeability barrier between the inducing metal and the sensor bacteria or because of complexation of the divalent cations with alginate. The adsorption of metal ions to alginate has been reported earlier for Ni (Lazaro et al., 2003), Cu and Co (Jang et al., 1995). It should be mentioned that in spite of poorer sensitivity of the bre-optic biosensors compared to non-immobilised bacteria the former still work in the environmentally relevant ranges: the usual concentrations of Hg, measured in natural sediments and soils are in g kg1 to mg kg1 range (Kannan et al., 1998; Norrstr m and Jacks, 1998). Moreover, the bre-optic bioseno sors have several advantages over non-immobilised bacteria, of which the main ones are ease of use of the system and possibility of in situ applications. 3.3. Reproducibility of the bre-optic biosensor assay Reproducibility of the bre-immobilised biosensor assay determined by variation of the results of 20 bres was 34%

(tested with 0.007 and 0.07 mg l1 of Hg2+ ), which was about 10 times poorer than the one obtained for non-immobilised Hg sensor E. coli MC1061 (pmerRluxCDABE), but still acceptable for a biological assay in eld conditions. 3.4. Storage of bre-optic biosensors The optimal storage conditions for bre-optic biosensor tips were seeked for, using Hg sensor as a model. Several storage media (LB, M9 and CaCl2 solution) and temperatures (80, 20, +4 C and ambient) were tested. The highest viability and sensitivity for target analyte was obtained when sensors were stored in CaCl2 solution at 80 C. In these conditions high performance of sensors was retained even after 2 months of storage (Fig. 3). The second and third best storage conditions were in LB at 80 C and in CaCl2 at ambient temperature. 3.5. Analysis of bioavailable Hg and As from natural soil and sediment samples The bre-optic biosensors were used for the analysis of bioavailable Hg and As in aqueous suspensions of 10 natural soils and sediments. The samples were relatively polluted (Table 2) as they originated from the Aznalcollar mining area, one of the main polymetallic deposits in Spain, which has caused environmental problems due to the release of mine tailings into the surroundings and contaminating the land and groundwater. The biosensor analysis of samples was made in parallel to the respective non-immobilised sensor bacteria and the results were compared. All the analysis were performed in water suspensions of the samples and thus, the sensor bacteria were theoretically exposed not only to the dissolved metals but also to particle-bound ones that may become bioavailable due to bacterial activity. It must be noted that the As sensor strain is inducible by As in both oxidation states, As(III) (arsenite) and As(V) (arsenate) (Table 1). The limit of quantication of the sensor for As3+ was about 10-fold lower than that for As5+ (Table 1), the most common As form under aerobic conditions in the nature

Fig. 3. The effect of storage on performance of Hg biosensor. Induction of bioluminescence by 0.1 mg l1 of HgCl2 ( ) and viability (expressed as colony forming units, CFU per bre tip) (columns) after storage in 0.15 M CaCl2 at 80 C.

A. Ivask et al. / Biosensors and Bioelectronics 22 (2007) 13961402 Table 2 Total and bioavailable amounts of Hg and As in the soil/sediment samples Samplesa Type Hg Bioavailable Totalb (mg kg1 ) GTS00683 GTS00684 GTS00685 GTS00686 GTS00687 GTS00688 GTS00689 GTS00690 GTS00691 GTS00692 Estonian PLV for industrial soilse Soil Soil Soil Soil Soil Soil Soil Soil Soil Sediment 11.7 <8 <8 <8 <8 <8 <8 <8 12.5 8.3 10 (mg kg1 ) (% of total) Fibre-optic Hg-biosensorc 0.019 (0.16) <0.013 0.075 <0.013 <0.013 <0.013 <0.013 <0.013 0.032 (0.26) 0.018 (0.22) As Bioavailable (mg kg1 ) (% of total) Totalb (mg kg1 ) 253 210 83 24 40 45 8 12 701 633 50 Non-immobilised As-sensord 7.4 (2.9) 13 (6.2) 1.8 (2.2) <0.8 1.1 (2.8) 2.2 (4.9) <0.8 <0.8 23 (3.3) 6.0 (0.9)

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Non-immobilised Hg-sensorc 0.78 (6.7) 0.005 0.6 0.0005 0.0006 0.0008 <0.0003 <0.0003 0.7 (5.6) 0.1 (1.2)

Fibre-optic As-biosensord 0.74 (0.29) 4.8 (2.3) <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 4.1 (0.6) 2.1 (0.3)

CV of bioavailability data did not exceed 40%. a Samples from Aznalcollar mining area in Spain, named by Grup de T` cniques de Separaci , Universitat Aut` noma de Barcelona during SENSPOL Technical e o o Meeting on Sensors for Characterisation and Monitoring of a Contaminated Site (69 November 2002, Sevilla, Spain) b Based on chemical analysis performed by Grup de T` cniques de Separaci , Universitat Aut` noma de Barcelona. e o o c E. coli MC1061 (pmerRluxCDABE). d E. coli MC1061 (parsluxCDABE). e PLV (permitted limit value) for industrial soils in Estonia determined by Pinnases ja p hjavees ohtlike ainete sisalduse piirnormid Keskkonnaministri m arus o a nr 12, 2 April 2002, RTL 40, 662.

(Matschullat, 2000). Because of the higher concentrations of As in the form of As5+ in the nature and higher limit of quantication (Table 1), As(V) was considered to be the main inducer of the sensor in natural samples and the calibration with As5+ was used to calculate the bioavailable As in the samples. 3.5.1. Analysis of bioavailable Hg 8 samples out of 10 induced the more sensitive Hg-biosensing system (not-immobilised sensor bacteria) whereas the less sensitive system (bre-optic biosensor) was induced in case of four samples (three of which contained Hg more than 8 mg kg1 that was also the limit of determination for the atomic spectroscopy under the conditions used in this study). The bioavailable fraction of Hg determined by non-immobilised bacteria varied between 1.2% and 6.7%. This is higher than the values reported by Pet nen and Romantschuk (2003), who analyzed the bioavaila ability of Hg in clayish soil by recombinant Hg sensor bacteria and showed that only 0.12% of Hg was bioavailable. These data again prove that the bioavailability (and thus, the direct hazard to biota) depends greatly on soil composition (texture, particle size, etc. (Alloway, 1995)). Indeed, in our previous study on bioavailability of methyl mercury sorbed to model soil components (montmorillonite, kaolinite, humic acid) and analyzed using E. coli MC1061 (pmerBRBS luc) we showed that the bioavailability of methyl mercury in different soil matrices ranged from 9% to 56% (Bernaus et al., 2005). As seen in Table 2, the bioavailable amount of Hg determined by the bre-optic Hg biosensor (0.2% of the total Hg in the soil as the average for three samples) was remarkably lower if compared to the results of non-immobilised bacteria. The lower apparent bioavailability of Hg for alginate-immobilised bacte-

ria in solid samples could be due to the insufcient contact of the immobilised bacteria with the solid matrix of the sample. Indeed, the contact with the solid matrix of soils or sediments is possible only for bacteria in outer layers and the bacteria in inner layers, contributing most to the response of the biosensor in terms of the light reaching the detector, are exposed only to the minor fraction of soluble metals. According to our previous studies less than 0.19% of the total soil Cd, Zn and Pb was leached to the water extracts (Kahru et al., 2005). The effect of the alginate matrix on the apparent bioavailability of Hg was also demonstrated by concentrationeffect curves for standard HgCl2 solution: the limit of quantication of the immobilised Hg sensors was about 100 times lower than for non-immobilised sensor cells (Table 1 and Fig. 2). Thus, when the environmental samples are analyzed, the results obtained by alginate-immobilised sensor bacteria may rather model the reduced bioavailability of metals to, e.g., biolm-entrapped micoorganisms. 3.5.2. Analysis of bioavailable As All the seven most arsenic-polluted samples (As level exceeding 40 mg kg1 ) induced the luminescence of non-immobilised sensor bacteria whereas only four of them (As level exceeding 210 mg kg1 ) induced the bre-optic arsenic sensor (Table 2). As was in the case of Hg the bioavailable fraction of As, detected with immobilised bacteria, was lower than the one determined with non-immobilised cells. However, compared to Hg, the differences between As bioavailabilities were smaller: the average difference in bioavailability in the four samples inducing both the non-immobilised bacteria and the bre-optic biosensor was fourfold. This result may indicate that the importance of particlecell contact in bioavailability was less signicant for As than for

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Hg. Moreover, in water solution As is obviously in form of oxyanion with negative net charge, which makes it less susceptible to binding to alginate matrix. These data are in accordance with the results obtained previously (Pet nen and Romantschuk, a 2003), showing that the bioavailable fraction of As in spiked soils was similar in soil-water suspensions and particle-free extracts. However, according to the results of Pet nen and Romantschuk a (2003) and the ones obtained in the current study, a considerable fraction of As (9599% of the total content in the samples analyzed in this study) remained bound to the solid particles and was not bioavailable even in case of direct contact between the solid matrix and bacteria. 4. Conclusions In this work, two luminescent bre-optic biosensors for the detection of Hg and As were developed. The biosensors consist of alginate-immobilised recombinant bacteria emitting light specically in the presence of bioavailable Hg or As in a dosedependent manner. The limits of quantication of the developed sensors (0.0026 mg l1 for Hg, 0.012 and 0.14 mg l1 for As(III) and As(V), respectively) are low enough to allow their use in environmental analysis. Storage at 80 C proved the most optimal, enabling alginate-immobilised bacteria used in bre-optic biosensor to maintain their viability and sensitivity towards analyte. The biosensors alongside with the non-immobilised Hg and As sensor bacteria were successfully applied for the analysis of bioavailable fractions of Hg and As in soil and sediment samples from the Aznalcollar mining area (Spain). About 20-fold more Hg and 4-fold more As was available to non-immobilised bacteria compared to the bre-attached bacteria. This was most probably due to the poorer contact of the alginate-immobilised bacteria with the sample. In the case of Hg, complexation of the Hg ions with the alginate matrix could also reduce the bioavailable fraction. The use of biosensors in the analysis of heavy metal pollution has several advantages: cost-effectiveness, simplicity and rapidity of the test procedure and little if any requirements for the sample pre-treatment. Moreover, differently from chemical methods they enable to measure the biologically available and thus, potentially hazardous fraction of metals. Considering all this, the biosensor analysis can successfully complement the analytical chemical methods in environmental risk assessment. Acknowledgements We would like to thank Kaisa Hakkila (University of Turku, Finland) for help and advice in practical work, Risto Juvonen

(Hidex Oy) for providing of some equipment and Margit Heinlaan for correcting of the manuscript. The study was partially funded by Maj and Tor Nessling Foundation, Academy of Finland (Grant Nos. 201232 and 207258), the Estonian Science Foundation (Grant No. 5551), the Israel Ministry of Science and the Arts, Grant No. 13139-1-98, the EC RD, PEBCAT and MENDOS project Contracts EVK1-CT-2000-00069 and QLRT2001-02323. T. Green and A. Ivask thank SENSPOL for travel support (SENSPOL Thematic Network, Contract No. EVK1CT1999-20001). References
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