Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue5.2000:
Soil Biology & Biochemistry 32 (2000) 639±646
www.elsevier.com/locate/soilbio
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 c
a
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 the mer-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 2.5 mg 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 the mer-lux biosensor is a useful and sensitive tool for estimation of bioavailable mercury in soil. 7 2000 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
* Corresponding author. Tel.: +45-3532-2053; fax: +45-35322040.
E-mail address: [email protected] (S.J. Sùrensen).
metals. Often the bioavailable fraction is de®ned as
being the solvent extractable fraction in the total concentration, 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 zooplankton and ®sh muscles has been used as indirect indicators of mercury bioavailability (Slotton et al.,
1995). Others used the total concentration of methylated mercury as a measure of bioavailability (Regnell
and Tunlid, 1991). None of the methods mentioned
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 9 0 - X
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L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 mercury, Hg(II), is the substrate for the formation of the
extremely toxic methyl-mercury, the most toxic mercury form that is accumulated and concentrated in the
foodchain (Barkay et al., 1992). In the environment
the rate of methylation is dependent on the concentration of Hg(II) (Compeau and Bartha, 1985; Gilmour 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 (luxCDABE ) quantitatively respond to Hg(II) (Selifonova
et al., 1993; Barkay et al., 1997). The mer promotor is
activated when Hg(II), present in the cytoplasm of the
biosensor bacterium, binds to MerR resulting in transcription of the lux genes and subsequent light emission. 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 community to mercury exposure in two dierent soils, an agricultural 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 density of cell suspensions in 67 mM phosphate buer
(pH 6.8) was adjusted to A660 corresponding to approximately 2 108 cells mlÿ1.
2.2. Soil samples and microcosm design
The soil used for the development of the soil merlux assay was collected from a garden farm in Kingston, TN, USA. Two dierent soil types were used in
microcosm experiments, an agricultural soil (no pesticides 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 evaporation. 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 HNO3 and 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. Experiments to optimise the period of shaking showed
decreased amounts of mercury in leachates when shaking exceeded 15 min (data not shown), and this shaking period was therefore chosen for all further
experiments. Large soil particles were removed from
leachates prior to assays by ®ltration or centrifugation.
Table 1
Soil characteristics
Total C %
Total N %
C/N ratio
Ammonium mg N/g dry soil
Nitrate mg N/g dry soil
Water holding capacity %
pH
Agricultural
Beech Forest
1.3
0.19
6.86
0.08
7.42
24
6.6
1.7
0.17
9.76
1.26
6.64
47
3.8
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
Filtrations were performed using acid rinsed millipore
®ltration setups. All ®lters used were nitro-cellulose ®lters (25 or 47 mm dia). Filtration through various poresizes, 0.22, 0.45, 0.8, 1.2, 3.0 and 8 mm, was
attempted. In another approach soil leachates were ®ltered through Watman ®lter paper (No. 40) placed in
glass funnels. Filtrates were collected in 300 ml Erlenmeyer ¯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 immediately transferred to the assay tubes. In microcosm
experiments centrifugation was used for the preparation 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 immediately 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 (Falcon, NJ, USA) for luminometer counting. The ®nal
assay medium consisted of pyruvate (5 mM); Na,Kphosphate buer (67 mM PO4; 34 mM Na; 33 mM K;
pH 6.8) and (NH4)2SO4 (91 mM). 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, Meriden, 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 regression between expression factors and mercury concentration 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) concentration and expression factors in the concentration
range of 0.3±1 nM (Rasmussen et al., 1997). Leachates
641
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 (Arizona 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 heterotrophs were enumerated on similar medium prepared
with 10 mg Hg(II) as HgCl2 mlÿ1. All plates were incubated at 24 8C for 4 d prior to enumeration.
2.7. Bacterial diversity
Diversity analysis of the microcosm bacterial community 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) (Muyzer 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. Following electrophoresis gel blocks containing the DNA
were cut from the gel and stored at ÿ208C in Eppendor 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 incubated for 20 min at 688C. PCR was performed with
`ready to go' PCR beads as described by the manufacturer (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 followed by 34 cycles of: 948C 1 min, 608C 1 min, 728C 1
min and the last cycle was followed by 8 min at 728C.
Dierent 16S rDNA sequences were separated using
DGGE as described by Muyzer et al. (1993). The D
GENE System equipment (BIO-RAD) was used for
the preparation of gels and electrophoresis. Gels were
stained with SYBR Green (1:10,000 dilution, Molecular Probes, Eugene, USA) for 1 h. The number of
bands in DGGE gels, counted manually from Polaroid
pictures were used as measure of bacterial diversity.
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L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 evaluate 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 inhibition of mer-lux induction with ®ltration through a
pore size smaller than 3.0 mm. The response in all ®ltrates was lower than that of the raw un®ltered leachate. 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 pressure released substances that reduced mercury bioavailability. 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 merlux 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 of mer-lux in soil (spiked with 1 mg Hg gÿ1 prior
to leaching). Leachates were ®ltered through ®lters with increasing pore size. (B) Induction of mer-lux in distilled water and a ®ltrate (0.22 mm)
of soil leachate spiked with 15 ng Hg(II) mlÿ1.
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
sponse that was nevertheless decreased relative to that
of un®ltered leachates (data not shown).
These results suggested that ®ltration by forcing leachates through nitro-cellulose ®lters might result in an
underestimation of bioavailable mercury concentrations.
The response of assays in leachate obtained by gravity ®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).
643
Gravity ®ltration was slow (approximate ¯ow rate
10 ml leachate in 60 min), making this procedure
extremely time consuming. Centrifugation was therefore used for soil leachate preparation in all following 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 mer-
Fig. 2. Comparison of biosensor response in gravity ®ltered, centrifuged and raw untreated soil leachates. Numbers in parenthesis are calculated
expression factors (see text for further information).
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L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
cury to the response of the soil microbial community
to mercury exposure in soil microcosms using two distinctly dierent soils.
Concentrations of bioavailable mercury were
measured over a period of 15 d after Hg addition.
Both soils demonstrated a decline in bioavailable mercury, albeit with dierent patterns (Fig. 3A). The initial 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 concentration of bioavailable mercury stayed just above
the detection limit (0.2 ng gÿ1) throughout the rest of
Fig. 3. The relationships of mercury bioavailability to the response of the soil microbial community. Microcosm experiments using an agricultural
(q) or a beech forest soil (*) were set up to relate bioavailable mercury (A) to the development of bacterial resistance to mercury measured as
percent Hg resistant bacteria of all culturable bacteria (B) and to bacterial diversity measured as number of 16S rDNA bands counted on
DGGE gels (C).
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 mercury (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 mercury 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 2.7 20.2 mg Hg gÿ1 dry soil on
d 0 and 2.6 2 0.1 mg Hg gÿ1 at the end of the experiment on d 15. During the same period, beech forest
soil had 3.220.2 and 3.320.5 mg Hg gÿ1 dry soil, respectively. The background concentration of total mercury was similar in both soils (0.19 and 0.27 mg 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 resistance 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 frequency remained, slightly above pre exposure quantities, 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 supplemented 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 provides 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 number on d 15. In the beech forest soil no eect of mercury exposure was observed on the number of 16S
rDNA bands, i.e. diversity, which were about 27 for
the duration of the experiment.
645
4. Discussion
This investigation showed that the mer-lux bioassay
is a useful tool for quanti®cation of bioavailable mercury 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 activity 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 bioavailable mercury in soil is water leachable.
Results of experiments designed to optimize the merlux bioassay in soil leachate showed the biosensor response might be biased by the presence of soil particles. 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 biosensor responses in assays performed on gravity ®ltered and centrifuged leachates with assays in raw
untreated leachate (Fig. 2). Assays performed with the
constitutive mutant showed that this was most probably due to shading by soil particles (Fig. 2B).
Little is known about the toxicity of mercury associated with colloidal and ®ne particles. Since these fractions 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 microbial response to mercury observed as development
of mercury-resistant bacteria and lowering of diversity
was correlated to changes in concentrations of bioavailable 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).
The heavy metal concentration at which a bacterial response is elicited may vary greatly depending on the
metal and the soil type (BaÊaÊth, 1989). In our study, the
dierences in response of the two bacterial communities
in the two soil types to dosing with equal amounts of
total mercury, con®rms that bioavailable rather than
total mercury is the factor controlling microbial responses. This is in good agreement with a study by Ran-
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L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 varied 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 ammonium (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 bioassay medium constituents have shown that the sensitivity 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 detectable concentrations in the soil, there was no enrichment of resistant bacteria or decline in community
diversity (Fig. 3). These results suggest that bioavailable 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 mercury is the substrate for mercury 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 processes in contaminated soil and sediment'' (www.biopro.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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for polymerase chain reaction ampli®cation and DNA ®ngerprint
analysis. Current Microbiology 29, 301±307.
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characteristics and cell location. FEMS Microbial Ecology 24,
321±331.
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Environmental Microbiology 63, 3291±3293.
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789±795.
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Microbiology 42, 593±603.
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for detection of bioavailable mercury(II) in the environment.
Applied and Environmental Microbiology 59, 3083±3090.
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patterns of biota in a seasonally anoxic northern California reservoir. Water Air and Soil Pollution 80, 841±850.
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www.elsevier.com/locate/soilbio
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 c
a
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 the mer-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 2.5 mg 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 the mer-lux biosensor is a useful and sensitive tool for estimation of bioavailable mercury in soil. 7 2000 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
* Corresponding author. Tel.: +45-3532-2053; fax: +45-35322040.
E-mail address: [email protected] (S.J. Sùrensen).
metals. Often the bioavailable fraction is de®ned as
being the solvent extractable fraction in the total concentration, 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 zooplankton and ®sh muscles has been used as indirect indicators of mercury bioavailability (Slotton et al.,
1995). Others used the total concentration of methylated mercury as a measure of bioavailability (Regnell
and Tunlid, 1991). None of the methods mentioned
0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 9 0 - X
640
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 mercury, Hg(II), is the substrate for the formation of the
extremely toxic methyl-mercury, the most toxic mercury form that is accumulated and concentrated in the
foodchain (Barkay et al., 1992). In the environment
the rate of methylation is dependent on the concentration of Hg(II) (Compeau and Bartha, 1985; Gilmour 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 (luxCDABE ) quantitatively respond to Hg(II) (Selifonova
et al., 1993; Barkay et al., 1997). The mer promotor is
activated when Hg(II), present in the cytoplasm of the
biosensor bacterium, binds to MerR resulting in transcription of the lux genes and subsequent light emission. 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 community to mercury exposure in two dierent soils, an agricultural 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 density of cell suspensions in 67 mM phosphate buer
(pH 6.8) was adjusted to A660 corresponding to approximately 2 108 cells mlÿ1.
2.2. Soil samples and microcosm design
The soil used for the development of the soil merlux assay was collected from a garden farm in Kingston, TN, USA. Two dierent soil types were used in
microcosm experiments, an agricultural soil (no pesticides 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 evaporation. 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 HNO3 and 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. Experiments to optimise the period of shaking showed
decreased amounts of mercury in leachates when shaking exceeded 15 min (data not shown), and this shaking period was therefore chosen for all further
experiments. Large soil particles were removed from
leachates prior to assays by ®ltration or centrifugation.
Table 1
Soil characteristics
Total C %
Total N %
C/N ratio
Ammonium mg N/g dry soil
Nitrate mg N/g dry soil
Water holding capacity %
pH
Agricultural
Beech Forest
1.3
0.19
6.86
0.08
7.42
24
6.6
1.7
0.17
9.76
1.26
6.64
47
3.8
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
Filtrations were performed using acid rinsed millipore
®ltration setups. All ®lters used were nitro-cellulose ®lters (25 or 47 mm dia). Filtration through various poresizes, 0.22, 0.45, 0.8, 1.2, 3.0 and 8 mm, was
attempted. In another approach soil leachates were ®ltered through Watman ®lter paper (No. 40) placed in
glass funnels. Filtrates were collected in 300 ml Erlenmeyer ¯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 immediately transferred to the assay tubes. In microcosm
experiments centrifugation was used for the preparation 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 immediately 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 (Falcon, NJ, USA) for luminometer counting. The ®nal
assay medium consisted of pyruvate (5 mM); Na,Kphosphate buer (67 mM PO4; 34 mM Na; 33 mM K;
pH 6.8) and (NH4)2SO4 (91 mM). 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, Meriden, 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 regression between expression factors and mercury concentration 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) concentration and expression factors in the concentration
range of 0.3±1 nM (Rasmussen et al., 1997). Leachates
641
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 (Arizona 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 heterotrophs were enumerated on similar medium prepared
with 10 mg Hg(II) as HgCl2 mlÿ1. All plates were incubated at 24 8C for 4 d prior to enumeration.
2.7. Bacterial diversity
Diversity analysis of the microcosm bacterial community 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) (Muyzer 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. Following electrophoresis gel blocks containing the DNA
were cut from the gel and stored at ÿ208C in Eppendor 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 incubated for 20 min at 688C. PCR was performed with
`ready to go' PCR beads as described by the manufacturer (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 followed by 34 cycles of: 948C 1 min, 608C 1 min, 728C 1
min and the last cycle was followed by 8 min at 728C.
Dierent 16S rDNA sequences were separated using
DGGE as described by Muyzer et al. (1993). The D
GENE System equipment (BIO-RAD) was used for
the preparation of gels and electrophoresis. Gels were
stained with SYBR Green (1:10,000 dilution, Molecular Probes, Eugene, USA) for 1 h. The number of
bands in DGGE gels, counted manually from Polaroid
pictures were used as measure of bacterial diversity.
642
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 evaluate 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 inhibition of mer-lux induction with ®ltration through a
pore size smaller than 3.0 mm. The response in all ®ltrates was lower than that of the raw un®ltered leachate. 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 pressure released substances that reduced mercury bioavailability. 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 merlux 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 of mer-lux in soil (spiked with 1 mg Hg gÿ1 prior
to leaching). Leachates were ®ltered through ®lters with increasing pore size. (B) Induction of mer-lux in distilled water and a ®ltrate (0.22 mm)
of soil leachate spiked with 15 ng Hg(II) mlÿ1.
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
sponse that was nevertheless decreased relative to that
of un®ltered leachates (data not shown).
These results suggested that ®ltration by forcing leachates through nitro-cellulose ®lters might result in an
underestimation of bioavailable mercury concentrations.
The response of assays in leachate obtained by gravity ®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).
643
Gravity ®ltration was slow (approximate ¯ow rate
10 ml leachate in 60 min), making this procedure
extremely time consuming. Centrifugation was therefore used for soil leachate preparation in all following 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 mer-
Fig. 2. Comparison of biosensor response in gravity ®ltered, centrifuged and raw untreated soil leachates. Numbers in parenthesis are calculated
expression factors (see text for further information).
644
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
cury to the response of the soil microbial community
to mercury exposure in soil microcosms using two distinctly dierent soils.
Concentrations of bioavailable mercury were
measured over a period of 15 d after Hg addition.
Both soils demonstrated a decline in bioavailable mercury, albeit with dierent patterns (Fig. 3A). The initial 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 concentration of bioavailable mercury stayed just above
the detection limit (0.2 ng gÿ1) throughout the rest of
Fig. 3. The relationships of mercury bioavailability to the response of the soil microbial community. Microcosm experiments using an agricultural
(q) or a beech forest soil (*) were set up to relate bioavailable mercury (A) to the development of bacterial resistance to mercury measured as
percent Hg resistant bacteria of all culturable bacteria (B) and to bacterial diversity measured as number of 16S rDNA bands counted on
DGGE gels (C).
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 mercury (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 mercury 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 2.7 20.2 mg Hg gÿ1 dry soil on
d 0 and 2.6 2 0.1 mg Hg gÿ1 at the end of the experiment on d 15. During the same period, beech forest
soil had 3.220.2 and 3.320.5 mg Hg gÿ1 dry soil, respectively. The background concentration of total mercury was similar in both soils (0.19 and 0.27 mg 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 resistance 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 frequency remained, slightly above pre exposure quantities, 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 supplemented 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 provides 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 number on d 15. In the beech forest soil no eect of mercury exposure was observed on the number of 16S
rDNA bands, i.e. diversity, which were about 27 for
the duration of the experiment.
645
4. Discussion
This investigation showed that the mer-lux bioassay
is a useful tool for quanti®cation of bioavailable mercury 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 activity 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 bioavailable mercury in soil is water leachable.
Results of experiments designed to optimize the merlux bioassay in soil leachate showed the biosensor response might be biased by the presence of soil particles. 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 biosensor responses in assays performed on gravity ®ltered and centrifuged leachates with assays in raw
untreated leachate (Fig. 2). Assays performed with the
constitutive mutant showed that this was most probably due to shading by soil particles (Fig. 2B).
Little is known about the toxicity of mercury associated with colloidal and ®ne particles. Since these fractions 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 microbial response to mercury observed as development
of mercury-resistant bacteria and lowering of diversity
was correlated to changes in concentrations of bioavailable 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).
The heavy metal concentration at which a bacterial response is elicited may vary greatly depending on the
metal and the soil type (BaÊaÊth, 1989). In our study, the
dierences in response of the two bacterial communities
in the two soil types to dosing with equal amounts of
total mercury, con®rms that bioavailable rather than
total mercury is the factor controlling microbial responses. This is in good agreement with a study by Ran-
646
L.D. Rasmussen et al. / Soil Biology & Biochemistry 32 (2000) 639±646
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 varied 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 ammonium (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 bioassay medium constituents have shown that the sensitivity 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 detectable concentrations in the soil, there was no enrichment of resistant bacteria or decline in community
diversity (Fig. 3). These results suggest that bioavailable 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 mercury is the substrate for mercury 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 processes in contaminated soil and sediment'' (www.biopro.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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