Journal of Environmental Biotechnology
Vol. 6, No. 2, 115–122, 2006

Original paper (regular paper)

Use of Plate-Wash Samples to Evaluate Bacterial Population
Dynamics in Mercury- and Trichloroethylene-Contaminated Soils
NOBUAKI MERA and KAZUHIRO IWASAKI*
National Institute for Environmental Studies, Tsukuba, Ibaraki 305-8506, Japan* TEL: +81-(0)298-50-2407 FAX: +81-(0)298-50-2407* E-mail: kiwasaki@nies.go.jp(Received; 5 September, 2006/Accepted; 30 October, 2006)
We used PCR-DGGE to analyze the eﬀects of mercury (HgCl2) and trichloroethylene (TCE) on soil bacterial communities, and we developed a method of using culturable bacteria to evaluate contaminated soils. Measurement of concentrationsof total bacteria in soils contaminated with HgCl2 (0, 1, 10 ppm) and TCE (0, 10, 100 ppm) revealed that bacterial concentrations remained the same regardless of type of pollutant, concentration, or sampling time. We then used PCR-DGGE to
analyze the community structures of total bacteria (soil samples) and culturable bacteria (plate wash samples). Changes inband patterns in HgCl2- and TCE-contaminated soil samples were small and similar, but in the plate wash samples there
were greater diﬀerences, and specific bands appeared. To determine relative changes in the bacterial communities, we analyzed the DGGE profiles by nonmetric multidimensional scaling. Over 200 days, changes in the bacterial communities were
small and similar in all soil samples from HgCl2-and TCE-contaminated soils. In contrast, plate wash samples of the same
soils showed changes in the bacterial communities; these changes were optimally detected after 50 days of culture. Unlike soil
sample analysis, analysis of plate wash samples eﬀectively clarified the eﬀects of HgCl2 and TCE on soil bacterial communities.
Key words: Soil bacteria, mercury, trichloroethylene, plate wash, PCR-DGGE

1.

Introduction

Since ancient times, natural ecosystems have stayed inharmony and balance with global material cycles (carbon,
nitrogen, sulfur) and energy flows. In recent years, however,various chemical substances, such as the volatile chlorinatedcompounds trichloroethylene (TCE)38) and tetrachloroethylene (PCE)27), the heavy metals mercury15) and lead26), andthe extremely stable polychlorinated biphenyls (PCBs)6) andpolychlorinated dibenzo-p-dioxins (PCDDs)12,25), were easily
discarded to the environment by industrial development,
and they have now become serious problems. These pollutants negatively aﬀect living organisms and the environment:
their accumulation in the environment confuses natural
ecosystems, and some pollutants show hormone-like actions
and carcinogenesis when they concentrate in the living body.
When nutrient sources become available in the naturalenvironment, not only activated bacteria but also dormant
and starvation forms of bacteria are swiftly activated to use
the nutrients as energy sources16). It is thought that where
various pollutants are available instead of nutrient sources
and specific bacteria using these pollutants lives in contaminated soils. For example, the TCE-degrading bacteria
Methylocystis sp. M33) and Mycobacterium sp. TA278); thePCE-degrading bacteria Desulfitobacterium sp. Y5129) andMethanosarcina sp. FR2); the mercury-reducing bacteria
Bacillus megaterium MB120) and recombinant mercury-

reducing bacteria Pseudomonas putida PpY101/pSR13410);
the PCB-degrading bacteria Pseudomonas pseudoalcaligenes KF70730) and Burkholderia sp. TSN10117); and the
PCDD-degrading bacteria Sphingomonas wittichii RW140)
and Pseudomonas resinovorans CA107) have been isolated,
and their metabolic pathways for the degradation of pollutants have been studied in detail.
In other studies of waste decomposition and the interaction of bacterial communities, various bacteria have beenfound to play important roles in the processes of decomposition in the natural environment and as field-scale composters19,23,24). If we are to eﬀectively utilize such bacteria forthe bioremediation of environmental pollutions, it is important that we (i) determine the characteristics of the specificbacteria that are predominate at contaminated sites; (ii)find out which bacteria coexist with these specific bacteria;and (iii) clarify the mechanisms of degradation of the pollutants at the sites of these bacterial communities. However,these pollutants consist of a number of chemical substances,and there are few data on their eﬀects on soil bacterial
communities. Recent studies have reported the eﬀects ofcoal tar components such as creosote37) and polycyclic aromatic hydrocarbons (PAHs)1); the eﬀects of petroleum contamination13); the eﬀects of the insecticide hexachlorocyclohexane (HCH) on microbial communities in contaminatedsoils22); and the eﬀects of PCB contamination on plant rhizospheres3). The results of basic information will increase

MERA et al.

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our understanding of the eﬀects of various pollutants on
soil bacterial communities.
In this study, we examined the eﬀects of inorganic mercury (HgCl2; a deadly heavy metal) and trichloroethylene (TCE;a carcinogen and an industrial pollutant of volatile chlorinated compounds) on the bacterial communities of soils
contaminated with these substances. We indicated the eﬀectiveness of using plate wash samples to analyze culturable
bacteria as indicators of the eﬀects of pollutants on soil
bacteria communities, and developed a method of evaluating contaminated soils by using the culturable bacteria.
Furthermore, using this method, we showed the possible toobtain the culturable and specific bacterial communities in
HgCl2- and TCE-contaminated soils.

2.
2.1.

Materials and methods

Soils, and preparation of soil microcosms, and soil
samples

We used alluvial soil taken from a field at the National
Institute for Environmental Studies, Tsukuba, Japan. The
initial water content of the soil was measured (26% [v/w]),
and the soil was air-dried to about 10% water content. The
dried soil was passed through a sieve (2-mm mesh) to makeit a uniform size. The water content of the soil was then adjusted to 20% by adding sterile distilled water, and the soil
was incubated at 10°C for 3 days in the dark. Soil microcosms were prepared in (i) 200-mL glass jars (AS ONE
Corp., Osaka, Japan), each containing 100 g (dry weight) of
the incubated soil and (ii) 69-mL vials (GL Sciences Inc.,Tokyo, Japan), each containing 10 g (dry weight) of the
incubated soil. The glass jar microcosms were contaminatedwith 0, 1, or 10 ppm of 2 mg/mL HgCl2, and the vials were
contaminated with 0, 10, or 100 ppm of 1100 mg/L TCE
saturated solution. To consume a vial every sampling because TCE is a volatile compound, a large number of TCEcontaminated microcosms were prepared. All microcosms
were adjusted to a 26% of final water content with sterile
distilled water and were incubated statically at 20°C in thedark. Each contaminated soil in microcosm was regularly
sampled and was used as a source of soil samples.
2.2.

Plate culture, bacterial cell counting, and plate
wash samples

Bacteria were cultured on R2A medium (yeast extract
0.5 g/L, proteose peptone 0.5 g/L, casamino acids 0.5 g/L,
dextrose 0.5 g/L, soluble starch 0.5 g/L, sodium pyruvate
0.3 g/L, potassium phosphate dibasic 0.3 g/L, magnesium
sulfate 0.05 g/L, agar 15 g/L, pH 7.2; Difco Laboratories,
Detroit, MI, USA) at 20°C for 1 week in the dark. The
number of culturable bacteria was counted by the plateculture method. Each contaminated soil sample (1 g wetweight) was dispersed in 9 mL of sterile distilled water andhomogenized by vortex at top speed for 3 min. The soil solutions obtained were decimally diluted, and 200 µL of eachdiluted solution was mixed into 2 mL of 0.7% (w/v) agarose

solution and swiftly spread onto an R2A agar plates. After
cultivation, the number of bacteria in the soil sample wascalculated from the number of colonies. Plate wash samples
were prepared as follows: Three ml of sterile distilled water
was poured on colonies on R2A agar plate, and all colonies
were mixed with a disposable inoculating needle. The cellsuspensions were used as the sources of plate wash samples.
2.3.

DNA extraction, and PCR amplification

Total bacterial DNA from 300-mg soil samples and culturable bacterial DNA from 200-µL plate wash samples
were extracted by using a Fast DNA Spin kit for soil (Qbiogene Inc., Carlsbad, CA, USA) in accordance with the manufacturer’s protocol. PCR was performed with universal
primers 350F-GC and 920R, located in the V3 region of the
16S rRNA genes18), using GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA) The PCR mixture
contained 10 µL of template DNA solution, 1×PCR Gold
buﬀer, 1.5 mM MgCl2, 200 µM dNTP (Roche Molecular
Systems, Branchburg, NJ, USA), 0.2 µM of each primer,
1.25 U AmpliTaq Gold (Roche Molecular Systems), andnuclease-free water (Promega Corp., Madison, WI, USA) ina final volume of 50 µL. Hot-start PCR was performed at
95°C for 10 min. The annealing temperature was initially
set at 60°C for and was then decreased by 1°C for every
two cycles for 15 s until it reached 50°C. Twenty additional
cycles were carried out at 50°C. Primer extension was
10 min at 72°C. After the reaction, the PCR products wereconfirmed by electrophoresis in 1% (w/v) agarose gels using
i-Mupid (Cosmo Bio Co., Tokyo, Japan).
2.4.

DGGE and data analysis

DGGE was performed by the D-Code universal mutation
detection system (Bio-Rad Laboratories, Hercules, CA,
USA) with 6% (w/v) polyacrylamide gels (acrylamide tobisacrylamide ratio, 37.5:1) with a 30% to 60% (w/v) denaturing gradient (100% was defined as 7 M urea and 40%[v/v] formamide) in 0.5×TAE (pH 8.0). Electrophoresis
was run at 60°C for 17 h at 70 V. After electrophoresis, thegels were stained at room temperature for 40 min with
Vistra Green (Amersham Biosciences, Buckinghamshire,UK) and scanned using Typhoon 9400 (Amersham Biosciences). DGGE gel images of band positions were digitizedwith ImageQuant software (Amersham Biosciences). Because bacteria can have multiple copies of their rRNA gene,care should be taken with the interpretation of the numberof species present in a DGGE band. Therefore, we refer toa band as a species rather than the number of bacteria. The
presence or absence of a band in each lane was converted
to a binary (1 or 0) matrix to make the data accessible tostatistical analysis35). From this binary matrix, a distancematrix (anti-similarity) was calculated21). Then, a distancematrix was analyzed by nonmetric multidimensional scaling(NMDS) with SPSS software (SPSS Japan Inc., Tokyo,
Japan). NMDS shows a map showing the relationships
among samples in a Euclidean plane such that very similar

Evaluation of bacterial communities by plate-wash samples

values were plotted close together. This graph of a Euclidean
plane is much easier to interpret than the original distancematrix. The axes (dimensions 1 and 2) in the map have no
special significance and can be rotated or mirrored without
influencing the relative distances between the points. Interpretation of NMDS map can be achieved by explaining
each dimension or by finding structures or patterns in
multidimensional space36). When it was applied to DGGE
data, the NMDS map shows every band pattern (a reflection of the community structure at a specific point in time)
as one point, and relative changes in community structure
could be visualized and interpreted by connecting consecutive points. NMDS had shown to be useful tool for analysis
of genetic structures34,36).

3.
3.1.

Results and Discussion

Numbers of bacteria in contaminated soils

Because distinguishing viable bacteria from dead bacteria
and mineral particles is diﬃcult, the exact numbers ofbacteria in soils are not yet known. In general, it is widely
recognized that several thousand millions of microorganisms exist in 1 g dry weight of environmental soils5).
The majority of soil bacteria are heterotrophs that requireorganic sources for their existence, but the concentrations ofthese organic sources in soils are very small. Most bacteria
have adapted themselves to the low concentrations oforganic sources in these severe environments. When soil
bacteria are cultivated, various and a large number ofbacteria are more cultivated in low-concentration broththan these of high-concentration broth9).
To reveal diﬀerences in the bacterial communities of
HgCl2- and TCE-contaminated soils, we therefore measuredchanges in the number of bacteria by the plate culture
method in R2A medium (low-nutrient broth). Figures 1A(Black circles, 0 ppm; Black squares, 1 ppm; Black triangles,
10 ppm) and 1B (White circles, 0 ppm; White squares,

117

10 ppm; White triangles, 100 ppm) show that contamination with HgCl2 or TCE had the same eﬀect on bacterial
numbers for sampling times, respectively. Furthermore, inboth cases changes in the pollutant concentration had noinfluence on bacterial numbers, which remained almost
constant for 200 days. In the case of HgCl2, we consideredthat mercury ion (Hg2+) might be strongly adsorbed onto
the surfaces of the soil components and that most of thesoil bacteria would be present in the interior spaces of thesoils particles. The soil adsorption characteristics for heavy
metals and various organic carbons are known39). In thecase of TCE, TCE contamination at 10 ppm and 100 ppm
may have been too low to influence the soil bacteria. On
the other hand, Jenkinson11) has reported that dead bacteria
are immediately digested by living bacteria for survival in
the soil environment. We assumed that the bacteria thatused the dead bacteria might increase in number and, consequently, that the apparent number of total bacteria might
be constant in contaminated soils. Furthermore, the plateculture method has some problems: for example, (i) only
those bacteria that are adapted to the broth and culture
conditions can be colony forming; (ii) some kinds of bacteria that are strongly influenced by the physical and chemical
eﬀects of the rapidly-formed colonies cannot form colonies;and (iii) some bacteria are extremely slow in proliferating.
3.2.

Eﬀects of HgCl2 and TCE on bacterial community
structure

Although the total concentrations of bacteria in HgCl2and TCE-contaminated soils were constant during the sampling times, we anticipated that the diversities of bacterial
communities in soil samples (included unculturable bacteria)
and in plate wash samples (culturable bacteria) would bechanged by the eﬀects of pollutants. PCR-DGGE was performed to evaluate the diversity of the bacterial communities using DNA solutions extracted from soil samples andfrom plate wash samples.

Fig. 1. Time courses of change in concentration of culturable bacteria in HgCl2- and TCE-contaminated soils.
The number of colony forming units of culturable bacteria per gram of soil was counted by the plate culture method. Soils were contaminated with various concentrations of HgCl2 and TCE. Diluted soil solutions were inoculated onto R2A agar plates and cultured at 20°C
for 1 week in the dark. After cultivation, the number of colonies was calculated. (A) Black circles, 0 ppm; black squares, 1 ppm; Black
triangles, 10 ppm; (B) White circles, 0 ppm; White squares, 10 ppm; White triangles, 100 ppm.

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MERA et al.

Fig. 2. DGGE analyses of diversity of total bacteria and culturable bacteria in HgCl2- and TCE-contaminated soils.
Every bacterial community was analyzed by PCR-DGGE using DNA solutions extracted from soil (total bacteria) and plate wash samples(culturable bacteria). A and B are results from soil samples, and C and D are results from plate wash samples; A and C were contaminatedwith HgCl2 and B and D with TCE. M, marker; P, positive control. (Positive controls in A and B were plate wash samples, and positive
controls in C and D were soil samples).

Figures 2A and 2B show samples of soils contaminatedwith HgCl2 or TCE, and Figures 2C and 2D shows plate
wash samples of the same soils. In the soil samples (total
bacteria), there were no sudden decreases in the number ofbands, and large numbers of bands appeared under contaminated conditions. The band patterns produced by bothcontaminants were similar. The eﬀects of changes in pollutant concentration on the bacterial community were verysmall, and the band patterns of the contaminated soils were
almost equal to those of the control (0 ppm) at the same
sampling times. In the plate wash samples (culturable bacteria), there were fewer bands than in the soil samples. Diﬀerences in the band patterns of HgCl2 and TCE contamination were apparent (Figs. 2C and 2D). The band patterns at
0 ppm and 1 ppm HgCl2 were similar. Two high-density
bands at 10 ppm (bands 1 and 2 in the figure) appeared at
all sampling times, and a specific band (band 3) appeared.
Although the band 1 at 0 ppm and 1 ppm (at 0 and
100 days) appeared, they decreased at 50 and 150 days, respectively. On the other hand, the specific band patterns
were shown at 10 ppm because the band 1 appeared at all
sampling times (for 0 to 200 days). The bacteria represented
by these bands (bands 1–3) may have been culturable mercury-resistant species. In the case of TCE, compared withthe control (0 ppm), changes in the band patterns at 10 ppm
and 100 ppm were increased at 50 days. High-density bands(bands 4 and 5 in the figure) appeared at 10 ppm, and spe-cific bands (bands 6 and 7) were found at 100 ppm. Furthermore, at 100 days there was a high-density band (band 8)
at 100 ppm. To notice the large change in band patterns of
TCE at 50 d, the bands 4 and 5 (at 10 ppm at 50 d), bands
6 and 7 (at 100 ppm at 50 d) were not shown at 50 day at
0 ppm although the band 5 (at 100 d), bands 7 and 8 (at150 d) appeared at 0 ppm TCE. Similarly, the band 8 (at100 ppm at 100 d) was not shown at 100 d at 0 ppm. It isvery interesting if these bands represented those bacteria
were TCE-degrading bacteria. Ellis et al.4) reported that the
total bacterial populations in the metal-contaminated soil
were largely unaﬀected by the metal inputs, and that themetal did not have a great eﬀect on diversity. As the total
bacterial diversity in soil is overwhelmingly high (more than
4000 species)32), the number of bands that was detected by
DGGE analysis was relatively low. Furthermore, it is known
that a single DGGE band does not always represent a single
bacterial strain28). Therefore, it is diﬃcult to discuss for
qualitative and quantitative results of band patterns andband density among soil samples from DGGE analysis, andwe considered that the changes in band patterns of soil
samples might appear to be very small, and that soil samples that included many kinds of bacteria would appear stable. These results indicated the eﬀectiveness of using plate
wash samples to analyze culturable bacteria as indicators of
the eﬀects of pollutants on bacterial communities.

Evaluation of bacterial communities by plate-wash samples

3.3.

Statistical analysis by NMDS

To reveal relative changes in the bacterial communities ofthe band patterns in the DGGE gels, we analyzed the
DGGE profiles by using NMDS. In NMDS analysis, theanti-similarities (diﬀerences) among bacterial communities
are calculated on the basis of diﬀerences in the band patterns and numbers of bands in the samples compared. Consequently, every bacterial community in the sample is shownas one point on a two-dimensional display. Relative changes
in the bacterial community can be shown by connecting theconsecutive points, and similarities in community structures
among samples can be indicated by the positions and distances of the points14,34,36).
Figures 3A and 3B show the relative change in the bacterial communities of the HgCl2- and TCE-contaminated soil,and Figures 3C and 3D show the plate wash samples. In thesoil samples (total bacteria), each movement of spot was
small and the large diﬀerences of spot movements were notshown among the pollutant concentrations (Figs. 3A and
3B). Although the DGGE band patterns of soil samples
were very similar (Figs. 2A and 2B), all spots at 0 days in
HgCl2 (0, 1, 10 ppm) and TCE (0, 10, 100 ppm) in NMDS
maps were shown large distance together. NMDS analyses,
constructs the relative map for bacterial communities. For

119these reasons, we considered that the movements of spots insoil samples might be relatively magnified within narrow
limits and these bacterial communities were therefore similar. On the other hand, the specific changes were shown in
the plate wash samples (culturable bacteria). The movements between the spots at 0 ppm and 1 ppm in the HgCl2
were small and the spots were relatively close together (Fig.
3C). In contrast, the positions of spots at 10 ppm were large
distances from that of the 0 ppm and 1 ppm. The movements between the spots for 0 to 50 days were especiallylarge at 10 ppm, the specific changes in the bacterial communities were indicated in this period. In the TCE, although
the time courses of spot movements were small at 0 ppm,
the spots at both 10 ppm and 100 ppm moved large distances for 0 to 50 days (Fig. 3D). As the each spot at
50-day was shown the reverse position, it was suggested thediﬀerences of bacterial communities between 10 ppm and100 ppm. To clear the time courses relative change in thebacterial communities of plate wash samples in detail,
NMDS was performed each contaminated concentration of
HgCl2 and TCE (Fig. 4). In the HgCl2, each movement ofspot at 0 ppm and 1 ppm was small over the 200-day sampling period, and all spots were generally close together (Figs.
4A and 4B). In contrast, the bacterial community at 10 ppm
changed greatly between 25and 100 days, because the spot

Fig. 3. NMDS analyses of relative changes in structure of bacterial communities in soil samples and plate wash samples from HgCl2- andTCE-contaminated soils.
Soil sample (total bacteria) results are shown as A and B, and plate wash samples (culturable bacteria) results are shown as C and D,
respectively. A and C were contaminated with HgCl2 (black and white circles, 0 ppm; black and white squares, 1 ppm; black and white
triangles, 10 ppm), and B and D with TCE (black and white circles, 0 ppm; black and white squares, 10 ppm; black and white triangles,
100 ppm). The number of days shown next to each spot is the sampling time.

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Fig. 4. NMDS analyses of relative changes in bacterial communities of culturable bacteria in HgCl2- and TCE-contaminated soils.
Every bacterial community was analyzed by NMDS. A, B and C in the upper row are results of plate wash samples from HgCl2-contaminated soils. D, E and F in the lower row are results of plate wash samples from TCE-contaminated soils. The number of days shown next
to each spot is the sampling time. Symbols: black circles, 0 ppm; black squares, 1 ppm; black triangles, 10 ppm; white circles, 0 ppm; whitesquares, 10 ppm; white triangles, 100 ppm.

movement was very large (Fig. 4C). Furthermore, the spots
at both 10 ppm and 100 ppm of the TCE moved large distances between 0 and 75 days (Figs. 4E and 4F). These results were consistent with Figures 3C and 3D, and indicatedlarge changes in the bacterial communities within this period. However, the spot movements between 0 and 25 days at
0 ppm were also large (Fig. 4D). As a reason for this phenomenon, we considered that because the microcosms containing TCE, which is volatile compound, were tightly shut
(0 ppm as control, 10 ppm, and 100 ppm), these airtightconditions might have aﬀected the bacterial communities.
These results suggested that the eﬀects of HgCl2 and TCE
on bacterial communities could be clarified by analyzing
plate wash samples taken from the microcosms after about
50 days of culture.
PCR-DGGE and NMDS analyses were performed on theplate wash samples to investigate the reproducibility of bacterial communities. We considered that there were various
bacteria in the uncontaminated control microcosms. Therefore, the control microcosm soils (0 ppm) were sampledindependently three times by the above-mentioned procedures. Every sample was cultured on three plates of R2A
medium (total of nine plates). The plate wash samples obtained were analyzed by PCR-DGGE and NMDS. Although
the densities of the bands in the DGGE gels diﬀered among
the samples, the band patterns were similar. Statistical analysis by NMDS revealed that there were no completely overlapping spots, but all the spots were gathered close together.

Therefore, the bacterial communities in all samples werehighly similar. Consequently, we confirmed a high rate of
reproducibility using this method of analysis of plate wash
samples (Data not shown). As the advantage of the plate
wash samples, all the bacteria in the plate wash samples canbe cultivated easily. Tamaki et al.31) used gellan gum as agelling reagent instead of agar to isolated novel microbes
from freshwater sediments; they reported that they were
performing continuous cultivation of these novel microbes
to reveal their physiological features and functional roles infreshwater sediments. We considered developing of detection systems for HgCl2- and TCE-contaminated soils using
the specific bacteria as an indicator, and to utilize for practical bioremediation using the degradable metabolism ofbacterial community.
In conclusion, we showed that the eﬀects of HgCl2 andTCE on bacterial communities could be clarified by the
analysis of plate wash samples (culturable bacteria) taken
after about 50 days of culture of the contaminated soil
microcosm. As the all bacteria in plate wash samples were
culturable bacteria, they should be isolated easily and it waspossible to use these bacteria for bioremediation of HgCl2and TCE-contaminated soils. We consider that the use ofthis method might be able to reveal the eﬀects of various
pollutants on bacterial communities in contaminated soils.
The use of plate wash samples is more eﬀective than soil
samples, because soil samples are apparently stable andincluded great kinds of bacteria.

Evaluation of bacterial communities by plate-wash samples

Acknowledgments
This work was supported in part by the Global Environment Research Fund.

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