Application of a
mer-lux
biosensor for estimating bioavailable
mercury in soil
Lasse D. Rasmussen
a, Sùren J. Sùrensen
a,*, Ralph R. Turner
b, Tamar Barkay
ca
Department of General Microbiology, University of Copenhagen, Sùlvgade 83H, DK-1307 Copenhagen K, Denmark b
Frontier Geosciences, Seattle, WA 98106, USA c
Deparment of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, NJ 08901-8525, USA
Accepted 1 October 1999
Abstract
A previously described bioassay using a mer-lux gene fusion for detection of bioavailable mercury was applied for the estimation of the bioavailable fraction of mercury in soil. The bioavailable fraction is de®ned here as being part of the water leachable fraction. Due to masking of light emission of soil particles leachates had to be cleaned prior to assays. Filtration of leachates through nitro-cellulose ®lters using pressure resulted in an underestimation of bioavailable mercury. Gravity ®ltration and centrifugation showed elevated (as compared with untreated leachate) and very similar responses. The utility of themer-lux
biosensor assay was tested by relating measurements of bioavailable and total mercury to the response of the soil microbial community to mercury exposure. Two dierent soil types (an agricultural and a beech forest soil) were spiked with 2.5mg Hg(II) gÿ1 in microcosms and the frequency of mercury resistant heterotrophs and changes in community diversity, de®ned as the number of dierent 16S rDNA bands observed in DGGE gels, were monitored. In the agricultural soil the initial concentration of bioavailable mercury was estimated to be 40 ng gÿ1
. This concentration did not change during the ®rst 3 d and coincided with increased degrees of resistance and a decrease in diversity. The concentration of bioavailable mercury decreased subsequently rapidly and remained just above the detection level (0.2 ng gÿ1
) for the remainder of the experiment. As a possible consequence of the decreased selection pressure of mercury, the resistance and diversity gradually returned to pre-exposure amounts. In the beech forest soil the concentration of bioavailable mercury was found to be about 20 ng gÿ1
throughout the experiment. This concentration did not at any time result in changes in resistance or diversity. This study showed that the bioassay using themer-luxbiosensor is a useful and sensitive tool for estimation of bioavailable mercury in soil.72000 Elsevier Science Ltd. All rights reserved.
1. Introduction
The most commonly used method for estimation of environmental risk due to heavy metal pollution is quanti®cation of total metals after digestion by strong acids and chemical analysis. However, this method gives little idea of the bioavailability of metals and their potential toxicity. Several investigators have attempted to measure the bioavailability of heavy
metals. Often the bioavailable fraction is de®ned as being the solvent extractable fraction in the total con-centration, e.g. weak acid (0.5 N HCl) extraction (Stone and Marsalek, 1996). Sterckerman et al. (1996) compared the concentration of metals extracted from soil using dierent solvents with concentrations in plants growing in the contaminated soils. They found close correlation between the water-extractable fraction and those taken up by plants. Accumulation in zoo-plankton and ®sh muscles has been used as indirect in-dicators of mercury bioavailability (Slotton et al., 1995). Others used the total concentration of methyl-ated mercury as a measure of bioavailability (Regnell and Tunlid, 1991). None of the methods mentioned
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* Corresponding author. Tel.: 2053; fax: +45-3532-2040.
above can account for the fraction of mercury that is available for microorganisms.
The issue of bioavailability is very critical with respect to mercury contamination. Bioavailable mer-cury, Hg(II), is the substrate for the formation of the extremely toxic methyl-mercury, the most toxic mer-cury form that is accumulated and concentrated in the foodchain (Barkay et al., 1992). In the environment the rate of methylation is dependent on the concen-tration of Hg(II) (Compeau and Bartha, 1985; Gil-mour et al., 1992). At present no analytical method can distinguish bioavailable forms of mercury.
Biosensors consisting of bacteria that contain gene
fusions between the regulatory region of the mer
operon (merR) and bacterial luminescence genes (
lux-CDABE) quantitatively respond to Hg(II) (Selifonova
et al., 1993; Barkay et al., 1997). The merpromotor is
activated when Hg(II), present in the cytoplasm of the biosensor bacterium, binds to MerR resulting in
tran-scription of the lux genes and subsequent light
emis-sion. This response is quantitative. At higher
concentration of Hg(II) mer promotors are activated
to a greater extent resulting in higher quantities of light emission. Rasmussen et al. (1997) improved the sensitivity of this bioassay to detect approximately 1.4
ng Hg(II) lÿ1 and the method has been applied for
measurements of bioavailable mercury in natural waters (Barkay et al., 1998; Turner et al., unpubl.). Our aim was to develop a method for the application of the bioassay to the measurement of bioavailable mercury in soil. The utility of this method was tested by relating measurements of bioavailable and total mercury to the response of the soil microbial commu-nity to mercury exposure in two dierent soils, an agri-cultural soil and a beech forest soil.
2. Materials and methods
2.1. Bacterial strains, plasmids, growth and cell preparation
The strains used were two mer-lux derivatives of
Escherichia coli HMS174, one containing plasmid pRB28 (Selifonova et al., 1993) and the other with a constitutive mutant of pRB28, pRB27 (Barkay et al., 1997). The constitutive mutant was used in all assays as a control to assure that light emission was not modulated by assay conditions (Barkay et al., 1997). Cultures were grown in LB medium using Kanamycin (Km) (50 mg mlÿ1
) for selection of plasmids. Growth
and preparation of cells for mer-lux assays were as
described by Selifonova et al. (1993). The optical den-sity of cell suspensions in 67 mM phosphate buer
(pH 6.8) was adjusted to A660 corresponding to
ap-proximately 2108cells mlÿ1.
2.2. Soil samples and microcosm design
The soil used for the development of the soil
mer-lux assay was collected from a garden farm in
King-ston, TN, USA. Two dierent soil types were used in microcosm experiments, an agricultural soil (no pesti-cides or fertiliser have been used for at least 20 yr) with crop change every year collected near Roskilde, Denmark and a beech forest soil from Grib Skov, Denmark (Table 1).
All soils were sieved (mesh size 2 mm) and air dried at room temperature over night. Water was added to
10% (v/w) of dry weight. Mercury as HgCl2 was
added to the soils with the water, water alone was added to control samples. Microcosm soils and the
garden soil were spiked with 2.5 and 1 mg Hg(II) gÿ1
soil, respectively. After addition of mercury and water the soils were placed in ziplock bags and mixed thoroughly by applying manual pressure to the outside of the bag. Samples were left at room temperature for 45 min prior to leaching. Three microcosms for each treatment consisting of 50 g soil, placed in 100 ml glass beakers in ziplock bags to minimise water
evap-oration. Microcosms were incubated at 248C. The
entire microcosm was transferred to a ziplock bag prior to each sampling, samples were obtained as described above and the remaining content of the bag returned to beakers. All glassware used were acid
rinsed using 2N HNO3and several volumes of distilled
water. Samples were collected from each of the three parallel microcosms at every sampling time.
2.3. Preparation of soil leachate
At least 1 g of soil (wet weight) was mixed with 10 volumes (w/v) of sterile double distilled water (ddH2O)
in a 300 ml Erlenmeyer ¯ask and the ¯ask was shaken horizontally at 300 rpm at room temperature. Exper-iments to optimise the period of shaking showed decreased amounts of mercury in leachates when ing exceeded 15 min (data not shown), and this shak-ing period was therefore chosen for all further experiments. Large soil particles were removed from leachates prior to assays by ®ltration or centrifugation.
Table 1
Ammoniummg N/g dry soil 0.08 1.26 Nitratemg N/g dry soil 7.42 6.64 Water holding capacity % 24 47
Filtrations were performed using acid rinsed millipore ®ltration setups. All ®lters used were nitro-cellulose ®l-ters (25 or 47 mm dia). Filtration through various
por-esizes, 0.22, 0.45, 0.8, 1.2, 3.0 and 8 mm, was
attempted. In another approach soil leachates were ®l-tered through Watman ®lter paper (No. 40) placed in glass funnels. Filtrates were collected in 300 ml Erlen-meyer ¯asks.
Soil leachates were centrifuged at 12,000g for 10 min
at 48C. Immediately after centrifugation or ®ltration
the cleaned leachate was transferred to acid rinsed scintillation vials, diluted with sterile ddH2O, and
im-mediately transferred to the assay tubes. In microcosm experiments centrifugation was used for the prep-aration of leachate.
2.4. Mer-lux assays in soil leachates
A concentrated mix (Barkay et al., 1998) of assay
constituants (180 ml) was added to soil leachates
im-mediately prior to assays. Assays were performed in 20 ml glass scintillation vials for detection of light by scintillation counting or in 6 ml polystyrene tubes (Fal-con, NJ, USA) for luminometer counting. The ®nal assay medium consisted of pyruvate (5 mM);
Na,K-phosphate buer (67 mM PO4; 34 mM Na; 33 mM K;
pH 6.8) and (NH4)2SO4(91mM). For quanti®cation of
bioavailable mercury, 1.72 ml of appropriate dilutions of soil leachate in water were added to a ®nal volume of 1,9 ml and assays were initiated by addition of 0.1 ml biosensor cell suspension (®nal concentration of 107 cells mlÿ1
). Light emission was recorded as either counts per min (cpm) in the single photon count mode of a Tri-Carp 2500 TR (Packard Instruments, Meri-den, CT) scintillation counter (counter setup: count time per sample 0.5 min, 20±30 cycles, no background correction, SPC %HV: 60); or Relative Light Units (RLU) per 30 s using a BG-P Portable luminometer (MGM instruments, Hamden, USA). Luminescence measurements were taken every 5±10 min for a period of 70±90 min.
2.5. Estimation of bioavailable and total mercury
The mer-lux expression factors (log quanta minÿ1) were calculated from the slopes of light emission curves as described by Barkay et al. (1998). A re-gression between expression factors and mercury
con-centration obtained from assays performed in ddH2O
containing known concentrations of Hg(II) was used to calculate bioavailable mercury concentrations in soil
leachates. Assays employed 107 cells of the biosensor
mlÿ1
to give a linear response between Hg(II) concen-tration and expression factors in the concenconcen-tration range of 0.3±1 nM (Rasmussen et al., 1997). Leachates
were diluted to give expression factors that fell within this concentration range.
Total mercury in soil microcosms was measured using a Jerome 431-x Mercury Vapor Analyser (Ari-zona Instruments, Phoenix, USA) using soil method 2 as described by Kriger and Turner (1995).
2.6. Enumeration of CFU
One g of soil was added to a test tube containing 9 ml 1% NaCl in dist. water and this suspension was vortexed at maximum velocity for 60 s. Appropriate 10-fold dilutions (0.1 ml) were plated on LB agar
plates containing the fungicide Natamycin 25 mg mlÿ1
(Merck) (Pedersen, 1992). Mercury-resistant hetero-trophs were enumerated on similar medium prepared with 10 mg Hg(II) as HgCl2mlÿ1. All plates were
incu-bated at 248C for 4 d prior to enumeration.
2.7. Bacterial diversity
Diversity analysis of the microcosm bacterial com-munity were performed at every sampling point by extracting total DNA, PCR ampli®cation of 16S rDNA fragments followed by sequence separation by denaturing gradient gel electrophoresis (DGGE) (Muy-zer et al., 1993). DNA extractions were carried out as described by Porteous et al. (1994), except that the sonication time was reduced from 3 min to 10 s since tests showed that DNA was rapidly lost with longer sonication time (data not shown). Removal of humic acids was performed by gel electrophoresis (0.7% low melting SeaPlaque agarose) for 1 h at 125 V. Follow-ing electrophoresis gel blocks containFollow-ing the DNA
were cut from the gel and stored at ÿ208C in
Eppen-dor tubes. Immediately prior to PCR ampli®cation
the gel blocks were melted at 688C for 5 min, 3
volumes of ddH2O were added and samples were
incu-bated for 20 min at 688C. PCR was performed with
`ready to go' PCR beads as described by the
manufac-turer (Pharmacia): 1 ml of DNA sample was mixed
with 675 nl of each primer (for primer sequence see
Muyzer et al. (1993)) and sterile ®ltered ddH2O to a
total volume of 25 ml. Ampli®cation was achieved by
one cycle of: 948C 4 min, 608C 1 min, 728C 1 min fol-lowed by 34 cycles of: 948C 1 min, 608C 1 min, 728C 1
min and the last cycle was followed by 8 min at 728C.
3. Results
3.1. Optimization of the mer-lux assay for the analysis of bioavailable mercury in soil leachate
In order to avoid blocking of light and changes in the concentration of bioavailable mercury during the assay, soil particles were removed from the leachates by ®ltration or centrifugation (see below). To evalu-ate the eect of ®ltration on the concentration of bioavailable mercury, leachates of the garden soil
(supplemented with 1 mg Hg(II) gÿ1 soil prior to
leaching) were ®ltered through ®lters with dierent pore sizes. The biosensor response declined with decreasing pore size (Fig. 1A) resulting in total
inhi-bition of mer-lux induction with ®ltration through a
pore size smaller than 3.0 mm. The response in all
®l-trates was lower than that of the raw un®ltered lea-chate. Thus, it seems that ®ltration sequestered
bioavailable mercury. This could be due to loss of mercury by binding to ®lters or soil particles collected on the ®lter or to the release of material that binds mercury during ®ltration. That the latter was the case
was shown by comparing induction in dist. H2O and
the 0.22 mm ®ltrate to which 0.75 nM Hg(II) (as
HgNO3) were added. The much lower response of the
leachate (Fig. 1B) indicates that ®ltration under press-ure released substances that reduced mercury bioavail-ability. Cell lysis that might have occurred during ®ltration is the likely source of these substances. This is supported by the fact that increasing pore size
resulted in less inhibition of mer-lux responses
(Fig. 1A) since the larger the pores the lower is the pressure built-up during ®ltration and the more likely are cells to pass intact through the ®lter. Indeed, when
pressure was varied during ®ltration induction of
mer-lux was totally abolished when high pressure was
applied while low pressure resulted in a signi®cant
re-Fig. 1. The eect of ®ltration of soil leachate on the bioavailability of mercury. (A) Induction ofmer-luxin soil (spiked with 1mg Hg gÿ1prior
sponse that was nevertheless decreased relative to that of un®ltered leachates (data not shown).
These results suggested that ®ltration by forcing lea-chates through nitro-cellulose ®lters might result in an underestimation of bioavailable mercury concentrations. The response of assays in leachate obtained by grav-ity ®ltrating through Watman ®lter paper (No. 40) was compared with leachate cleaned by centrifugation.
Both treatments showed elevated and very similar responses (Fig. 2A). Expression factors indicating the rate of increase in light emission (Barkay et al., 1998) were calculated to be 0.119 and 0.112 for
gravity ®ltration and centrifugation respectively.
While that of the untreated leachate was only 0.081. These results con®rm that soil particles had to be removed to prevent underestimation of the amount of bioavailable mercury in soil leachates.
Assays using the constitutive mer-lux derivative
HMS174/pRB27, revealed that underestimation was due to masking of light in raw leachate (Fig. 2B).
Gravity ®ltration was slow (approximate ¯ow rate 10 ml leachate in 60 min), making this procedure extremely time consuming. Centrifugation was there-fore used for soil leachate preparation in all follow-ing experiments. Furthermore, since centrifugation also removed indigenous bacteria from the leachate and previous work (Rasmussen et al., 1997) showed that bacterial density in the assay medium aect the assay's sensitivity, this method was favored over gravity ®ltration.
The constitutive mutant derivative of the biosensor,
strain E. coli HMS174/pRB27 (Barkay et al., 1997)
showed that light emission was not quenched in any of the soil leachates employed here (data not shown).
3.2. Microcosm experiments
3.2.1. Bioavailable and total mercury
The utility of the mer-lux biosensor was tested by
relating measurements of bioavailable and total
cury to the response of the soil microbial community to mercury exposure in soil microcosms using two dis-tinctly dierent soils.
Concentrations of bioavailable mercury were
measured over a period of 15 d after Hg addition. Both soils demonstrated a decline in bioavailable mer-cury, albeit with dierent patterns (Fig. 3A). The in-itial decrease that is apparent between d 0 and d 1 after spiking might have been attributed to an
overesti-mation of bioavailable mercury on d 0 due to the fact that an equilibrium between mercury and soil binding sites may not yet have been established after 45 min. After this initial decrease the agricultural soil retained a constant concentration of bioavailable mercury of
about 40 ng gÿ1
soil, followed by a considerable decrease to 0.3 ng gÿ1
between d 3 and d 5. The con-centration of bioavailable mercury stayed just above the detection limit (0.2 ng gÿ1
) throughout the rest of
the experiment. This decrease in bioavailable mercury concentration was not observed in the beech forest soil
in which about 20 ng Hg gÿ1
soil was found for the duration of the experiment (15 d; Fig. 3A). In both soils only a very small fraction of the total added mer-cury (2.5 mg gÿ1) was bioavailable and the transition from available to unavailable forms was rapid with only 2% available Hg an hour after addition of mer-cury to the soil (Fig. 3A).
The concentration of total mercury did not change in the soils during the experiment. In the agricultural soil total mercury was 2.720.2 mg Hg gÿ1dry soil on d 0 and 2.620.1 mg Hg gÿ1
at the end of the exper-iment on d 15. During the same period, beech forest soil had 3.220.2 and 3.320.5 mg Hg gÿ1
dry soil, re-spectively. The background concentration of total mer-cury was similar in both soils (0.19 and 0.27mg gÿ1
in agricultural and beech forest soil, respectively), the cause of the dierent concentrations of mercury after addition is not known.
3.2.2. Frequency of mercury resistant bacteria
In the agricultural soil the amount of mercury resist-ance began to increase after the ®rst day of exposure. The frequency of mercury-resistant isolates increased
rapidly reaching its peak on d 4 at 7.122% (Fig. 3B).
A gradual decline followed until a plateau was reached between d 7 and d 11, where the % Hg resistance fre-quency remained, slightly above pre exposure quan-tities, for the duration of the experiment.
No eect of the added mercury was observed on the frequency of mercury resistant bacteria in the beech forest soil with a constant fraction of approx. 0.004% of the total CFU growing on the mercury sup-plemented medium (Fig. 3B).
3.2.3. Bacterial diversity
Diversity was evaluated by the number of bands visualised in DGGE gels after electrophoresis of PCR ampli®cation product obtained using primers speci®c to all eubacterial 16S rRNA genes. This analysis pro-vides information on the diversity of the eubacterial community at large (i.e. without the need to culture soil bacteria) (Muyzer et al., 1993; Heuer and Smalla, 1997).
Diversity of the indigenous bacteria in the two soils responded dierently to mercury exposure (Fig. 3C). In the agricultural soil diversity decreased rapidly in the ®rst 4 d of the experiment with the average number of 16S rDNA bands declining from 26 to 20. After this minimum the number of bands gradually increased for the rest of the experiment reaching the initial num-ber on d 15. In the beech forest soil no eect of mer-cury exposure was observed on the number of 16S rDNA bands, i.e. diversity, which were about 27 for the duration of the experiment.
4. Discussion
This investigation showed that the mer-luxbioassay
is a useful tool for quanti®cation of bioavailable mer-cury in soil leachate obtained from three dierent soils. In this study the water-leachable fraction of mercury is referred to as being potentially bioavailable. This is in agreement with Sterckeman et al. (1996) who found a strong correlation between water-extractable mercury and accumulation of mercury in plants. Bacterial ac-tivity in the soil is located in niches characterized by the availability of water, and the fact that Hg(II) needs to be in aqueous solution in order to be transported to the cytoplasm is supporting the assumption that bioa-vailable mercury in soil is water leachable.
Results of experiments designed to optimize the
mer-lux bioassay in soil leachate showed the biosensor
re-sponse might be biased by the presence of soil par-ticles. Forced ®ltration to remove soil particles, resulted in an underestimation of bioavailable mercury (Fig. 1A). Assays performed in water and ®ltrate spiked with 0.75 nM Hg(II) showed that this was due to mercury-binding ligands that were released during ®ltration, probably by pressure-induced cell lysis (Fig. 1B). This corresponds well with earlier ®ndings that dissolved organic carbon (DOC) considerably decreases biosensor response (Barkay et al., 1997). That removal of soil particles from leachates was needed to avoid underestimation of the concentration of bioavailable mercury was shown by comparing bio-sensor responses in assays performed on gravity ®l-tered and centrifuged leachates with assays in raw untreated leachate (Fig. 2). Assays performed with the constitutive mutant showed that this was most prob-ably due to shading by soil particles (Fig. 2B).
Little is known about the toxicity of mercury associ-ated with colloidal and ®ne particles. Since these frac-tions of the total mercury will be eliminated from the soil leachate by centrifugation eventual bioavailability of these will not be detected by this assay.
The microcosm experiment showed that the mi-crobial response to mercury observed as development of mercury-resistant bacteria and lowering of diversity was correlated to changes in concentrations of bioa-vailable mercury (Fig. 3). Both these responses indicate toxicity and are well documented eects of heavy metal contaminations in soil (Roane and Kellogg, 1996; Ranjard et al., 1997; Smit et al., 1998).
Ran-jard et al. (1997) who after spiking four dierent soil types with the same mercury concentration found that after incubation the proportion of resistant bacteria var-ied from 0.4 to 36% . Many abiotic factors may aect the bioavailability of heavy metals in soil e.g. clay content, pH, dissolved organic carbon, root exudates (BaÊaÊth, 1989; Barkay et al., 1997; Giller et al., 1998). The main dierences in the soil variables (Table 1) between the agricultural and the beech forest soil is pH and
am-monium (NH4) content. Soil pH is often found to have
a large in¯uence on metal availability due to its strong eect on metal solubility and speciation. Thus, a decrease in pH usually results in increased availability (Giller et al., 1998). This seems not to be the case in this study where the pH in the beech forest soil was almost 3 units lower than that in agricultural soil (Table 1). The high ammonium content in the beech forest soil (Table 1) may be an important factor decreasing bioavailability of mercury. Experiments with varying the concentration of the dierent bioas-say medium constituents have shown that the sensi-tivity of the assay is decreased by increasing the ammonia concentration (unpublished data).
The results from beech forest soil shows that although bioavailable mercury was present in detect-able concentrations in the soil, there was no enrich-ment of resistant bacteria or decline in community diversity (Fig. 3). These results suggest that bioavail-able mercury at the detected concentration may not have been toxic to indigenous bacteria in beech forest soil. The bioassay requires the presence of bioavailable Hg(II) in the biosensor cytoplasm. This species of mer-cury is the substrate for mermer-cury methylation by soil bacteria (Beckert et al., 1974). The fact that detectable bioavailable mercury in beech forest soil was not toxic suggests that a potential for mercury methylation exists at subtoxic concentrations of mercury.
Acknowledgements
L.D.R. is supported by ``Centre for biological pro-cesses in contaminated soil and sediment'' (www.bio-pro.dk) under The Danish Environmental research programme. The authors wish to thank Pia Kringelum and Inge E. Larsen for excellent technical assistance.
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