FEMS Microbiology Letters 204 (2001) 49^53
www.fems-microbiology.org
Adaptation of the bacterial community to mercury contamination
Anne Kirstine Mu«ller, Lasse Dam Rasmussen, SÖren Johannes SÖrensen *
Department of General Microbiology, University of Copenhagen, SÖlvgade 83 H, DK-1307 Copenhagen K, Denmark
Received 25 April 2001 ; received in revised form 31 July 2001; accepted 1 August 2001
First published online 14 September 2001
Abstract
The utilisation of 31 sole carbon sources by bacterial communities of soil in the presence of increasing concentrations of Hg(II) was
measured by a colour development assay. The assay was performed on Biolog microtitre plates (Ecoplates) in the presence of Hg(II) and
compared to Hg(II)-free Ecoplates. Furthermore, community tolerance to Hg(II) was measured by colour development in microtitre plates
supplemented with LB broth and by enumeration of colony-forming units on LB agar plates. Both microtitre plates supplemented with LB
and LB agar plates contained increasing concentrations of Hg(II). The difference in substrate utilisation profile, as shown by growth on 31
different carbon substrates in the Ecoplates, suggested an adaptation of the soil community that correlated with the metal exposure level in
the soil. Similarly, growth on microtitre plates supplemented with LB and plate-spreading data showed an increased community tolerance
with increasing levels of mercury in the soil. Both the multi-function microtitre plate assay (Ecoplate) and the LB broth microtitre plate
assay are suitable for evaluating the adaptation of the bacterial community in soil to a heavy metal pollutant. ß 2001 Published by
Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
Keywords : Mercury; Community tolerance ; Ecoplate
1. Introduction
Many parameters are used to indicate the e¡ects of
heavy metals on the soil microbial community. E¡ects
can be measured by the size of the microbial biomass
and/or by the abundance of speci¢c organisms and di¡erent microbial-mediated processes [1,2]. However, such
measurements do not always indicate the direct e¡ects of
heavy metals on the microbial community as they fail to
account for the impact of other factors such as soil pH
and organic matter content. A property speci¢cally connected with the heavy metal concentration in the soil is the
tolerance of the microbial community to the compound of
interest. As the heavy metal exerts a selection pressure
upon the micro-organisms, a measurable increase in community tolerance will result.
Analysis of plate-spreading results have shown the proportion of heavy metal-resistant bacteria in soil to increase
with increasing concentration of the metal [3^5]. A resistance index determined by plate count has been proposed
* Corresponding author. Tel.: +45 35 32 20 53; Fax: +45 35 32 20 40.
E-mail address : [email protected] (S.J. SÖrensen).
as a relevant parameter when evaluating the e¡ect of
heavy metals [6]. Measurement of [3 H]thymidine incorporation into bacteria in the presence of increasing levels of
heavy metal has also been used to show increased tolerance of the soil bacterial community with increasing concentration of metal in the soil [7].
The increased tolerance of the community may also result in changes in the functional performance. Monitoring
functional changes in microbial communities by the sole
carbon source utilisation pro¢le has previously been used
to study the e¡ect of Hg(II) in soil [8,9] and community
tolerance to Zn(II) [10].
The aim of this research was to develop a rapid multifunction test for evaluating the tolerance of soil bacterial
communities to a speci¢c pollutant.
Soil was treated with four di¡erent concentrations of
mercury. After 1 week the adaptation of the bacterial
communities was measured by the growth kinetics in microtitre plates containing di¡erent mercury concentrations.
Growth on microtitre plates where each well contains one
of 31 di¡erent carbon substrates (Ecoplates) revealed the
sole carbon source utilisation pro¢les in the presence of
Hg(II) and Hg(II)-free controls. Furthermore, the number
of colony-forming units (CFU) grown on LB agar plates
with increasing levels of mercury was recorded.
0378-1097 / 01 / $20.00 ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 7 6 - 7
FEMSLE 10124 17-10-01
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2. Materials and methods
2.1. Soil microcosms
In autumn 1999 soil samples were collected from the
upper 20 cm of an agricultural soil on Zealand, Denmark
(soil characteristics : [11]). The soil was mixed, air-dried
overnight at room temperature and sieved (2 mm) before
being transferred to four boxes (50 g in each). The mercury was added to the soil as HgCl2 dissolved in water to a
¢nal concentration of 0, 2.5, 10 and 25 Wg Hg(II) g31 soil
and a water content of 15%. The soil was incubated in
dark, closed plastic boxes for 1 week at 25³C. Prior to
sampling the soil was mixed well.
2.2. Mercury-tolerant CFU
For bacterial counts (CFU) 1 g of soil was diluted in
9 ml phosphate-bu¡ered saline [8] and vortexed at maximum velocity for 1 min. Dilutions were made and 100 Wl
were spread on 10% LB (10U dilution of standard LB) [8]
(Merck, Darmstadt, Germany) agar plates supplemented
with fungicide (25 Wg natamycin ml31 ) [8] and 0, 0.1, 0.2,
0.4, 0.8, 1.0, 2.5 or 10 Wg Hg(II) ml31 . The number of
CFU was recorded after 4 days of incubation at 25³C.
The minimum inhibitory concentration (MIC) was determined by the lowest dilution plate displaying no colonies.
Note : 10% LB was used only in experiments involving
enumeration of CFU. In all other cases, the standard concentration of LB was used.
2.3. Microtitre plates
In the study reported herein we used two types of microtitre plates: Ecoplates and a no-substrate microtitre
plate (Biolog, Hayward, CA, USA). The Ecoplate contains
3U31 di¡erent sole carbon sources together with a tetrazolium redox dye, whereas the substrate-absent microtitre
plates contain only the tetrazolium redox dye. The formation of purple colour (measured by the absorbance of light
at 590 nm) will take place when microbial respiration reduces the dye.
Substrate-free microtitre plates were prepared by adding
15 Wl sterile LB broth to each well (¢nal volume : 150 Wl
per well). The plates were allowed to dry in a sterile air
£ush and prior to analysis, Hg(II) was added to the wells
as 10-Wl aliquots in H2 O to a ¢nal concentration of 0^10.0
Wg Hg (II) ml31 . Colour development was observed in
triplicate at each mercury concentration.
Microtitre plates containing three replicate wells of 31
di¡erent carbon substrates and one control well containing
no substrate were used for determining the metabolic activities of the four soils in the presence of 0 or 1 Wg Hg(II)
ml31 .
All microtitre plates were inoculated with 140 Wl of a
1033 dilution of the soil suspension prepared for enumer-
ation of tolerant CFU. The total number of cells in the
inoculum was evaluated by acridine orange direct count.
The microtitre plates were incubated in the dark and at
room temperature for 4^5 days. Colour development was
measured by light absorbed at 590 nm (OD590 nm ) every 4^
8 h with a microplate reader (EL 340 Biokinetics Reader,
Biotek Instruments, USA) and the data collected and analysed using KC4 (Bio-Tek Instruments).
2.4. Analysis of microtitre plate data
The maximum rate of colour development within wells
was calculated using light absorbance readings taken at
four time intervals during the period of incubation. The
time required to achieve the maximum rate of colour development was also recorded. A positive result was recorded when the maximum rate was s 0.006 OD590 nm
h31 as it was only in these wells that the cells were regarded as being su¤ciently metabolically active with respect to oxidation of substrate. The MIC of mercury in
the microtitre plates supplemented with LB was calculated
as the lowest concentration where no well recorded a positive result.
Measurement of colour development on Ecoplates led
to the recording of the number of positive wells, the average maximum rate of colour development and the average
time before obtaining the maximum rate of colour development for all the positive wells. Furthermore, the maximum rate of colour development for each substrate was
analysed by principal component analysis based on the
correlation matrix using SPSS 6.1 for Macintosh.
3. Results and discussion
As expected, the tolerance of the microbial community
increased with increasing levels of mercury in the soil. This
was indicated by the results obtained by all three methods
used.
By use of plate counting, the MIC was shown to increase with increasing concentrations of mercury in the
soil (Table 1). Furthermore, the number of colonies able
to grow in the presence of mercury increased with increasing levels of mercury in the soil (Fig. 1A).
Table 1
MIC of mercury for CFU and colour development in microtitre plates
containing LB
Hg(II) in soila (Wg g31 )
0
2.5
10
25
a
MIC Hg(II) (Wg ml31 )
CFU
Colour development
in microtitre plates
1
2.5
10
s 10
0.8
2.5
10
10
Microcosm soil exposed to four levels of mercury.
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51
small di¡erences in the total number of bacteria between
the four soils (3.3U108 ^5.9U108 g31 soil). As this was the
case, the wells were inoculated with the same dilution from
all soils (3.3^5.9U105 cells ml31 ).
Sterility of the pre¢lled microtitre plates was con¢rmed
as no colour development occurred when inoculated with
sterile water instead of soil suspensions. Also, as there was
no colour development in the wells containing sterile water
and Hg(II), mercury does not cause false-positive readings
as found with zinc [12]. Finally, cell lysis in the presence of
Hg(II) did not lead to an added source of nutrients as
there was no colour development in wells containing no
substrate but soil suspension and Hg(II).
The measurements of the maximum rate of colour development in the wells supported the results obtained by
Fig. 1. A: CFU in soil incubated with 0 (open bars), 2.5 (light grey
bars), 10 (dark grey bars) and 25 (black bars) Wg Hg(II) g 31 soil. The
CFU were grown in 10% LB medium [8] with increasing concentrations
of mercury. B: The maximum rate of colour development. C: The time
before reaching the maximum rate of colour development in microtitre
plates containing LB and increasing concentrations of mercury for the
same soils. Error bars indicate S.E.M. of three replicates.
Similar results upon exposure of other metals to soils
have been reported. The MIC of copper for growth of
aerobic soil bacteria in soils amended with manure containing copper was found to be greater than the MIC
recorded for reference soils [4]. Another study has shown
the percentage of bacteria resistant to cadmium or zinc to
increase with increasing levels of contaminating cadmium
or zinc in the soil [3]. In other work involving the investigation of cadmium- or zinc-contaminated soils, the ratio
of sensitive to resistant bacteria was lower compared to
reference soils [6].
It is important to know the original number of cells
used to inoculate the wells in the microtitre plates as any
resulting colour development is dependent on this cell
number. Total cell number as estimated by acridine orange
direct count and by enumeration of CFU indicated only
Fig. 2. A: The number of positive wells (maximum rate of colour development s 0.006 OD590 nm h31 ) B: The average maximum rate of colour
development for positive wells. C: The average time until achieving the
maximum rate of colour development for positive wells in microtitre
plates containing 31 di¡erent sole carbon substrates (Ecoplates) inoculated with soil incubated with 0, 2.5, 10 and 25 Wg Hg(II) g31 soil.
Open bars represent Ecoplates without Hg(II); black bars represent
Ecoplates with 1 Wg Hg(II) ml31 in the wells. Error bars indicate
S.E.M. of three replicates.
FEMSLE 10124 17-10-01
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A.K. Mu«ller et al. / FEMS Microbiology Letters 204 (2001) 49^53
enumeration of CFU (Fig. 1B). With increasing mercury
concentration in the wells, the maximum rate of colour
development decreased and the time before reaching the
maximum rate increased (Fig. 1C). This was especially
apparent in the soil containing 0 or 2.5Wg Hg(II) g31
soil. The MICs (Table 1) estimated by observations of
colour development in microtitre plates supplemented
with LB were also very similar to the results obtained by
enumeration of CFU. No colour development was observed in the wells containing 0.8 Wg Hg(II) ml31 when
inoculated with Hg(II)-free soil. However, when soil was
incubated with 10 and 25 Wg Hg(II) g31 soil, colour development was observed in wells containing 2.5 Wg Hg(II)
ml31 . Maximum rates of colour development of 28%
and 47% of the rate measured in Hg(II)-free wells were
recorded, respectively.
The results demonstrate the validity of the use of microtitre plates for evaluating community tolerance. Furthermore, additional parameters can be obtained by observations of colour development (indicative of cellular
respiration) on microtitre plates. The maximum rate of
colour development and the length of the time before
the maximum rate was achieved were measured. Both parameters correlated with the level of metal in the soil.
The number of utilised substrates (maximum rate of
colour development s 0.006 OD590 nm h31 ) in the
Hg(II)-free microtitre plates containing 31 di¡erent carbon
substrates (Ecoplates) was consistent at around 20 for all
four soils (Fig. 2A), while the functional response of the
community to the addition of mercury in the wells varied
between soils. Addition of Hg(II) resulted in a reduction
of the number of wells recorded positive upon incubation
with all soils but the number of positive readings increased
as the Hg(II) concentration increased in the soil. The average maximum rate of colour development was reduced
and the average time before reaching the maximum rate
was increased only in soils containing 0 and 2.5 Wg Hg(II)
g31 soil in the Ecoplates containing Hg(II) compared to
the Hg(II)-free Ecoplates (Fig. 2B,C).
In the principal component analysis on whole-plate
maximum rate of colour development (Fig. 3). The ¢rst
principal component (PC1) explained 41.1% of the total
variance and separated the Ecoplates with and without
mercury for all four soils. The level of similarity between
substrate utilisation pro¢les in the presence and absence of
Hg(II) correlated with the pre-exposed Hg(II) concentration in the soil. The results show that the mercury-adapted
bacterial community maintains its functional ability under
further exposure to mercury.
The results also show that the functional performance
of the community was altered in the presence of mercury
as the second principal component (PC2) separated the
soils according to level of mercury in the soil. These results
con¢rm previous ¢ndings that sole carbon source substrate
utilisation can detect functional changes in communities
exposed to environmental stress [8,9,13].
Fig. 3. Principal component analysis performed on the maximum rate
of colour development for each of the 31 sole carbon substrates in
microtitre plates (Ecoplates) inoculated with soil incubated with
0 (squares), 2.5 (circles), 10 (diamonds) and 25 (triangles) Hg(II) g31 soil.
Open symbols represent Ecoplates without mercury in the wells; ¢lled
symbols represent Ecoplates with 1 Wg Hg(II) ml31 in the wells. The
percentage of the total variation explained by the principal components
1 and 2 is 41.1% and 17.4%, respectively.
The ideal methods would evaluate the tolerance of the
total microbial community, but this is di¤cult to achieve.
The enumeration of tolerant CFU is selective since only a
minor fraction of the bacterial community is able to grow
on the nutrient-rich agar plates even at lower nutrient
levels.
As for enumeration of tolerant CFU, the microtitre
plate method is also selective since the coloration depends
on bacterial respiration on the substrate in the wells [14].
The utilisation of substrate can be attributed to more than
one bacterial type, but it does not have to be quantitatively related to the numbers of utilising bacteria [15]. The
level of adaptation of the most tolerant bacteria can explain the MIC values, and the maximum colour development rate re£ects the rate of metabolism of these organisms at the incubation condition.
It is possible that a larger proportion of the soil bacteria
are able to grow in the wells as opposed to on agar plates,
as in wells they do not have to make colonies on a solid
surface. The results from the two methods where the same
growth medium (10% LB) was used are very similar. It
therefore seems reasonable to assume that more or less
the same part of the community has been investigated.
The study of metal tolerance of the community for
many di¡erent functional activities hopefully increases
the fraction of bacteria included in the assay when compared to plating on one medium containing relatively high
amounts of nutrients.
The degree of utilisation of speci¢c substrates could give
ecologically relevant information if it was possible to select
more relevant substrates for the microtitre plates. The
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ability of resistant bacteria to degrade aromatic compounds has been found to be less than that of sensitive
organisms [6]. We did not ¢nd the same traits in our experiment.
In conclusion, we have found that the tolerance of the
community evaluated by the use of microtitre plates where
wells contain one of 31 di¡erent carbon substrates (Ecoplates) or LB broth is in accordance with the tolerance
evaluated by the traditional plate counting method. Both
the multi-function microtitre plate assay and the microtitre
plates containing complex media are simple and easy to
handle and o¡er rapid techniques to evaluate the tolerance
of the community in soil to a pollutant or other substances
of interest. The time scale of this experiment was 1 week as
the aim was only to demonstrate the validity of the method. We believe this will be a valuable method to detect
changes in the tolerance of the microbial community after
long-term exposure.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
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