MICROBIAL

ECOLOGY
Microb Ecol (2000) 40:177–188
DOI: 10.1007/s002480000033
© 2000 Springer-Verlag New York Inc.

Analysis of Microbial Communities in a Landfill Leachate

Polluted Aquifer using a New Method for Anaerobic
Physiological Profiling and 16S rDNA Based Fingerprinting

W.F.M. Röling,1 B.M. van Breukelen,2 M. Braster,1 M.T. Goeltom,1,* J. Groen,2

H.W. van Verseveld1
1
Section Molecular Microbial Ecology, Department of Molecular Cell Physiology, Faculty of Biology,
Research School SENSE Vrije Universiteit, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands
2
Department of Hydrology, Faculty of Earth Sciences, Vrije Universiteit, De Boelelaan 1085, NL-1081 HV
Amsterdam, The Netherlands

Received: 3 January 2000; Accepted: 21 March 2000; Online Publication: 28 August 2000

A B S T R A C T
Databases containing information regarding presence and activity of microbial communities will be
very useful for determination of the potential for intrinsic bioremediation in landfill leachate
polluted aquifers. Simple analyses such as community-level physiological profiling (CLPP) and
denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments yield large sets of data for
inclusion into such databases. In this study we describe the development of a method for anaerobic
CLPP, using commercially available Biolog plates. Incubation at the in situ temperature of the
aquifer (10°C) for 28 days was optimal for obtaining a specific, reproducible physiological profile.
Anaerobic incubation was essential for profiling anaerobic communities. The anaerobic cultivation-
dependent CLPP method and cultivation-independent DGGE were applied to groundwater and
sediment samples from the aquifer near the Coupépolder landfill in The Netherlands. A combina-
tion of computer-assisted CLPP and DGGE analysis of both groundwater and sediment samples
yielded the best separating power for characterizing microbial communities in the aquifer. Com-
munities in groundwater were significantly different from those in the corresponding sediment.
Microbial communities present in subsamples from sediment cores usually were similar for the
various sampling locations. Variation was observed for the heterogeneous sediment beneath the
landfill. Both anaerobic CLPP and DGGE analysis clearly separated microbial communities from the
polluted aquifer underneath the landfill from those in the less or not polluted aquifer downstream
and upstream of the landfill.

* Present address: Inter University Research Center (IURC) for Biotechnol-

ogy, Environmental Biotechnology Division, Technology Institute of Band-
ung, JI. Ganesha no. 10, Bandung, Indonesia.
Correspondence to: Dr. Wilfred F.M. Röling; Fax: +31 20 4447229; E-mail:
reuling@bio.vu.nl
178
Introduction
Landfilling has a profound effect on the environment as
effluent rich in organic matter can leach into underlying
aquifers. The originally oligotrophic aquifers develop into
relatively copiotrophic environments. Because of limited
availability and replenishment of electron acceptors, a se-
quence of redox zones will develop [7]. Old municipal land-
fills present a major risk to the environment as toxic material
may also leach into underlying aquifers and pollute them. As
such, landfills demand considerable attention. Present day
society aims at removing threats from landfills, although the
costs associated with traditional measures, such as pump and
treat, are extremely high. For The Netherlands, it has been
estimated that remediation of its 4,000 old landfills will cost
about 10 billion Euro.
However, research on landfill plumes has shown that the
spreading of pollution in aquifers is often less than expected,
due to natural attenuation [7]. Recently, it was shown that
the composition of the microbial community at petroleum
polluted aquifers was indicative for the occurrence of intrin-
sic bioremediation of benzene [23]. Composition of micro-
bial communities could also be indicative of (potential for)
intrinsic bioremediation at landfill leachate polluted aqui-
fers.
A database linking data regarding composition of micro-
bial communities, redox situation, and other hydrogeo-
chemical parameters to potential for intrinsic bioremedia-
tion and allowing rapid and inexpensive determination of
potential for intrinsic bioremediation at presumed hazard-
ous landfills would be very useful. Data regarding the com-
position of microbial communities preferably should be ob-
tained via simple, information-rich profiling methods. A
suitable method could be denaturing gradient gel electro-
phoresis (DGGE). The separation of PCR fragments of the
bacteria-specific 16S rDNA gene in a denaturing gradient
results in cultivation independent profiling of dominant
members in microbial communities [15, 22]. Profiles can
easily be compared by using standardized electrophoresis
conditions and computer-assisted pattern analysis [9, 10]. A
disadvantage of 16S rDNA based molecular techniques is
that these methods do not give information about the physi-
ological capacities of the microbial community. A rapid
method for obtaining physiological profiles of microbial
communities was developed by Garland and Mills [13] and
is based on the use of commercially available 96-well Biolog
(Biolog Inc., Hayward) microtiter plates that contain 95 dif-
W.F.M. Röling et al.
ferent substrates and the color redox dye tetrazolium violet.
This community-level physiological profiling (CLPP) ap-
proach has been shown to be very useful for comparing
communities in environmental samples [12, 19].
Therefore, both DGGE and CLPP could be suitable and
complementary methods to easily obtain a large data set
regarding microbial communities in aquifers beneath land-
fills. However, so far CLPP has only been performed under
aerobic conditions, whereas aquifers underneath landfills are
usually anaerobic [7], thus, a relevant physiological profile
can only be obtained by using an anaerobic method. In this
paper we describe the development of an anaerobic method
for CLPP. As a test of its suitability for profiling microbial
communities at landfill leachate polluted landfills, we ap-
plied the anaerobic CLPP method and DGGE to sediment
and groundwater samples obtained from the aquifer up-
stream, beneath, and downstream of the Coupépolder land-
fill, Alphen aan de Rijn, The Netherlands. The body of this
landfill is contaminated with benzene, toluene, ethylbenzene
and xylene, leaking into the underlying aquifer.
Site Description and Hydrogeology
The former Coupépolder landfill is situated in a polder area
near Alphen aan de Rijn, The Netherlands. The landfill has
an area of 22 ha. The southern part of the landfill is sepa-
rated from a 40 m thick Pleistocene sand aquifer by a Ho-
locene aquitard, consisting of a 6–10 m thick, heavy marine
clay layer, with generally a 1 m thick peat layer at the bottom
and a thicker peat layer at the top. In the northern part of the
landfill the clay layer, peat, and part of the aquifer have been
eroded by a tidal channel system during the late Holocene.
The channel deposits are very heterogeneous and consist of
fine layered sands, silts, and sandy clays. The Pleistocene
aquifer consists of moderately fine to coarse sands with some
intercalations of gravel and loam. The difference in hydraulic
head between the landfill (at 0 m mean sea level (MSL)) and
the aquifer (at −3.20 to −3.70 m MSL) causes a downward
flow toward the aquifer [25]. The flow takes place through
the channel deposits rather than through the less permeable
massive clay layer, as indicated by chloride measurements. In
the aquifer the leachate moves to the northeast along with
the regional flow, at about 30 m/year, and is expected to stay
in the upper part [24]. Therefore, sediment and groundwa-
ter sampling was performed in the channel deposits and
upper part of the Holocene aquifer.
Community Profiling of Polluted Aquifers 179

a
Table 1. Codes, locations and depths of groundwater samples and sediment cores taken at Coupépolder landfill

Depth (m -surface) Depth (m -surface)

Sample code Date of sampling Location sediment core groundwater sample

T (Begemann) 27-05-1997 Channel deposits 6–7.45 —

UP 15-10-1997 100 m upstream 10.5–11.2 15
A 21-10-1997 Channel deposits 9.6–10.3 10
B 29-10-1997 30 m downstream 10.0–10.7 14–15
C 29-10-1997 320 m downstream 10.8–11.5 15
a
From the sediment cores of locations UP, B, and C, two samples were taken for analysis; for sediment core from location A, four samples; see Fig. 1.

Materials and Methods sampling tube by applying nitrogen pressure on the pipe. Ground-
water for microbiological analyses was collected in glass bottles by
Field Sampling and Sample Treatment
allowing the bottles to overflow. Bottles were capped with as little
A sediment sample for determining the optimal conditions for the headspace as possible remaining and immediately transferred to the
anaerobic Biolog technique was collected at the transition of the laboratory. After storage at 4°C, the next day bottles were trans-
landfill with the underlying channel deposits (location T; Table 1)
using a Begemann sampler (Geotechnics Delft, The Netherlands).
This technique is capable of taking continuous and undisturbed
samples by pushing the sample into a nylon stocking. The sample
was transported in an anaerobic Plexiglas tube to the laboratory
and sampled under nitrogen atmosphere in an anaerobic glove box.
Samples of the core were stored in an anaerobic jar at 4°C.
Sediment cores for geochemical characterization were anaero-
bically collected with a core pushing device ([2]; Delft Geotechnics,
The Netherlands) at locations UP, A, B and C (Table 1). The
sampling depth was chosen using information about the geology
obtained by performing cone penetration tests in advance. After
retrieving the core (length: 70 cm; internal diameter: 68 mm) the
ends were immediately sealed with paraffin under continuous
flushing with nitrogen gas. Capped cores were placed in an anaero-
bic Fe(II)SO4 bath (1.7 g L-1 FeSO4⭈7H2O, 3.0 g L-1 ammonium
acetate). Cores were stored at 4°C for less than 24 h before they
were sampled under nitrogen atmosphere in an anaerobic glovebox
(Mecaplex) or glovebag (Instruments for Research and Industry,
Cheltenham). The outer ends of the cores (several centimeters)
were not used. From the sampled upstream (UP) and downstream
(B, C) cores smaller samples (subsamples) were taken at approxi-
mately one-third and two-thirds of the core length. These sub-
samples are indicated in figures and text with extension “-sed1” and
“-sed2,” respectively, after their location code. As depicted in Fig. 1,
from the core retrieved beneath the landfill (A), three subsamples
were taken, and also a mixture of the whole core was used. For
Biolog experiments, sediment was put into a small anaerobic glass
bottle and immediately transferred to an anaerobic glovebox for
analysis. For molecular analysis, sediment was frozen at -80°C until
DNA isolation.
At locations UP and B groundwater samples were recovered
from existing PVC piezometers, using a peristaltic pump. At loca- Fig. 1. Schematic cross-sectional overview of core A taken from
tions A and C groundwater samples were taken from an iron pipe the channel deposits underneath the landfill. Depicted are the ma-
with a 10-cm stainless steel screen driven to the desired depth terial descriptions, the depth below surface and the samples taken
(Delft Geotechnics, The Netherlands). Water in the pipe was kept from the core for microbial analysis (A-sed1, A-sed2, A-sed3, and
under anaerobic conditions and pushed upward through a Teflon mixture A-sedMix).
180
ferred to an anaerobic glove box for Biolog analysis. After that, 100
ml samples were vacuum filtrated over 45 mm diameter 0.2 µm
filters (Sartorius). Filters were stored at -80°C until isolation of
DNA.
Groundwater and Sediment Analysis
Oxygen, pH, and electrical conductivity were measured in the field
by electrodes placed in flow cells. Groundwater samples were ana-
lyzed for chloride, dissolved organic carbon (DOC), nitrate, iron,
manganese, sulfate, sulfide, methane, BTEX, and common chlori-
nated aliphatics. Sampling and analysis were performed by a com-
mercial laboratory (IWACO BV, Rotterdam, The Netherlands) ac-
cording to Dutch standard norms.
Grain size distribution of sediment was measured on a Laser
Particle Sizer A22 (Fritsch, Idar Oberstein, Germany) and used to
calculate the clay and silt fraction [18].
Characteristics of Color Development in Biolog Plates
Color development under nongrowing conditions was studied by
growing Escherichia coli JM109 in mineral M9 medium [3] con-
taining 0.4% glucose, at 35°C. In the exponential stage (OD660
0.2–0.3) either rifampicin or chlorampenicol was added to a final
concentration of respectively 20 or 150 µg/ml. After 1 h extended
incubation, cells were centrifuged and washed with 0.85% NaCl.
Serial dilutions in 0.85% NaCl were prepared up to 10-9 and in-
oculated into Biolog plates; antibiotic was added to all solutions. A
culture without antibiotics added in any of the steps was used as
control. Plates were incubated up to 3 weeks in plastic bags, to
avoid evaporation, and were examined daily for color development.
Anaerobic Community-Level Physiological Profiling
The procedure was set up based on recommendations by Biolog
Inc. for the identification of anaerobic bacteria [5]. Anaerobic Bio-
log GN plates (Biolog Inc., Hayward CA, USA) were prepared by
placing them into 2.5 L anaerobic jars (Workshop Vrije Universi-
teit), evacuating and refilling with anaerobic gas (95/5% N2/CO2).
This process was repeated three times. Anaerobic profiling experi-
ments were performed in a glove box (Mecaplex, Grenchen, Swit-
zerland) containing a hydrogen-free 95/5% N2/CO2 anaerobic gas
mixture. Preparation of anaerobic plates and placement of plastic
materials (4 h dry heat sterilized at 120°C) into the glove box was
performed at least 1 day in advance of an experiment, in order to
allow traces of oxygen to diffuse out of the materials. Anaerobic
extraction solution (0.1% sodium pyrophosphate) and suspension
solution (0.4% NaCl, 0.086% NaHCO3) was prepared by addition
of oxygen scavenger (Na2S.9H2O) and redox indicator (methylene
green) to concentrations of 0.002% and 0.00015%, respectively,
from 1000× stock solutions after sterilization (20 min, 120°C) and
cooling in an anaerobic atmosphere. Solutions were allowed to
equilibrate for at least 1 day. Tenfold dilutions of groundwater in
suspension medium were prepared. For sediment samples, 2.8 g of
W.F.M. Röling et al.
sediment was aseptically added to a 28 ml bottle that was then filled
with extraction medium. The bottle was wrist shaken for 2 min,
after which large particles were allowed to settle for 30 s. Tenfold
dilutions were prepared in suspension medium. Appropriate dilu-
tions were inoculated into Biolog plates at 150 µl per well. Plates
were wrapped in plastic bags and placed in anaerobic jars together
with a hydrogen-free anaerobic generating system (Anaerogen, Ox-
oid, Bastingstoke, England). Substrate utilization during incubation
was scored after transferring the jars to the glove box and visually
inspecting the plates. Anaerobiosis of the jar was checked by re-
moving the Anaerogen sachet from the jar and subjecting it to
oxygen. If the sachet became warm it was assumed that the jar was
still anaerobic.
For the numerical comparison and clustering of the data, the
programs Bionumerics 1.1 and GelCompar 4.0 (Applied Maths,
Kortrijk, Belgium) were used. Substrates not utilized in any of the
plates in an experiment were excluded from analysis. Simple
matching coefficient was then used for calculation of similarity; this
coefficient is calculated as Sm = nAB/ntotal, in which nAB is the
number of substrates that in plate A as well as in plate B are both
positive or negative. ntotal is the total number of substrates in the
plates. The resulting matrix was subjected to UPGMA (unweighted
pair group method using arithmetic averages) clustering.
DGGE Profiling
DNA extraction was performed according to Duarte et al. [8].
Direct extractions from sediment were started by mixing 10 g of
sediment with 4.5 g glass beads, 6 ml 120 mM sodium phosphate
(pH 8.0), 0.75 ml 10% SDS, and 5 ml phenol (pH 8.0) and per-
forming bead beating in a Braun homogenizer at 3400 rpm. Filters
containing groundwater microorganisms were cut in small pieces
and put in a bead beater tube to which 0.8 ml 120 mM sodium
phosphate buffer (pH 8.0), 0.6 g glass beads (0.1 mm diameter),
100 µl 20% SDS, and 0.7 ml phenol (pH 8.0) was added. A bead
beater (BioSpec Products, Techno Lab, Alkmaar, The Netherlands)
was used at 4200 rpm. DNA was purified by one or two rounds of
Wizard column purification (Promega, Madison, WI). DNA from
sediment was first purified by one round of Sepharose 4B purifi-
cation [17]. PCR were performed in a total volume of 25 µl con-
taining 0.4 µM primer F357-GC [21], 0.4 µM primer R518 [21], 0.4
mM dNTPs, 10 µg BSA, Expand buffer (Boehringer, Mannheim,
Germany), 2.6 U Expand enzyme, and 1 µl undiluted DNA tem-
plate. Amplification was performed in a Perkin Elmer DNA
Thermo Cycler as follows: 94°C for 4 min, after which 35 cycles of
94°C for 0.5 min, 54°C for 1 min, and 72°C for 1 min, with a final
elongation at 72°C of 5 min.
DGGE was performed with the Bio-Rad DCode system. PCR
product was loaded on 1 mm thick 8% (wt/vol) polyacrylamide
(37.5:1 acrylamide:bisacrylamide) gels containing a 40–60% linear
denaturing gradient; 100% denaturant is defined as 7 M urea and
40% (v/v) formamide. Gels were run in 1× TAE buffer (16 mM
Tris-HCl (pH 8.0), 0.8 mM Na-EDTA, 20 mM acetic acid) at 70 V
for 16 h. Gels were stained in 1× TAE buffer containing 1 µg ml-1
ethidium bromide and recorded with a CCD camera system (The
Community Profiling of Polluted Aquifers
Imager, Appligen, Illkirch, France). Gel images were converted,
normalized, and analyzed with the GelCompar 4.0 software pack-
age (Applied Maths, Kortrijk, Belgium), using Pearson product-
moment or Jaccard coefficient and UPGMA clustering. To aid the
conversion and normalization of gels, a marker consisting of DNA
from culturable microorganisms on green vanilla beans was added
on the outside of the gel as well as after every four samples. The
outer two lanes of the gels were not used. For analysis using Jaccard
coefficient a band position tolerance of 0.7% was applied. This was
the minimum tolerance at which all marker lanes clustered at
100%.
Results
Development of Anaerobic Community-Level Physiological
Profiling Method
A sediment sample obtained from just below Coupépolder
landfill (Table 1; location T) was used to develop an anaero-
bic community-level physiological profiling (CLPP) method.
The sediment was anaerobic; oxygen was below the detec-
tion limit (<0.1 mg L-1).
To avoid any influence of oxygen, all treatments were
performed under anaerobic conditions and solutions were
made anaerobic by the addition of an oxygen scavenger.
Initially, sodium thioglycolate was used at 150 mg L-1 as
oxygen scavenger. However, since this is a rather unnatural
and carbon-containing substance, it was replaced by sodium
sulfide. A concentration of 20 mg L-1 Na2S⭈9H2O was suf-
ficient to remove oxygen without causing direct reduction
by sulfide of the tetrazolium in Biolog GN plates. False posi-
tive reactions occurred at 40 mg L-1 or more. No influence
of type of oxygen scavenger on the physiological profile was
observed. When microorganisms in samples were either re-
moved via filtration with a 0.2 µm filter or inactivated by
adding mercury chloride to a final concentration of 0.5 g L-1,
no color development occurred, demonstrating that the pro-
files observed are due to microbial activities.
To establish optimal incubation conditions, the effects of
temperature, length of incubation, and dilution were exam-
ined. A clear influence of incubation temperature was ob-
served when plates inoculated with the same suspension
were incubated at the in situ Coupépolder groundwater tem-
perature of 10°C, room temperature (20–22°C), and the gen-
erally used temperature for cultivation, 37°C (Fig. 2A). Sub-
strates were not significantly utilized at 37°C. Although the
total number of substrates utilized after 31 days were similar,
development of number of positive wells was faster at 10°C
than at room temperature. Similarity (using simple match-
ing) after 31 days of incubation was 78%, while duplicate
181
plates incubated both at 10°C were 94% similar after a com-
parable incubation time (Fig. 2C). Thus, incubation tem-
perature has an obvious influence on rate of development of
positive wells and physiological profile. Therefore, the in situ
temperature of the aquifer was chosen as the incubation
temperature in the next experiments.
Optimum incubation time and reproducibility were de-
termined for two independently prepared suspensions from
the same sediment sample, diluted 1:100 (1 g sediment
added to 100 ml suspension medium). Figure 2B shows that
a rapid increase in positive wells occurred during the first
week of incubation, but after 4 weeks of incubation only
minor changes occurred. Similarities between duplicate
samples were calculated using simple matching on all 95
substrates or via disregarding in the similarity calculation
substrates that were not utilized within the data set (in this
case equaling the use of the Jaccard coefficient for correla-
tion). In the case of low numbers of positive wells, as after a
few days of incubation, simple matching based on all 95
substrates automatically led to high similarities (Fig. 2C).
Disregarding substrates that are not utilized gave a more
realistic picture and showed that the plates were more than
90% similar after >14 days incubation. Also, in other inde-
pendent experiments similarities above 90% were found be-
tween duplicate plates after 28 days (see also Fig. 3). Con-
sequently, the method is reproducible and an incubation
time of 4 weeks is sufficient. Substrates that are not utilized
within an experiment should not be included in cluster
analysis.
Sediment and groundwater have low total cell counts,
about 106–10-1 per g and 105–106 per ml respectively [4, 16,
20]. Although a single cell per Biolog-well is sufficient to
reduce the tetrazolium dye in the wells due to growth [11],
only a minor part of the microbial community likely will be
culturable [1]. In order to maximize the number of positive
wells and avoid diluting out species, which will affect the
reproducibility, the effect of dilution was determined. The
use of 1:10 dilutions for sediment samples often led to high
backgrounds and sometimes reduced tetrazolium, even in
the control well with water only. A similar observation was
also made for undiluted groundwater. A 1:10 dilution for
groundwater samples or 1:100 dilution for sediment did not
give a background color in the control. Further dilution of
the sediment samples delayed the increase in the number of
substrates utilized; the final number of positive wells after 4
weeks was slightly less than for the 1:100 dilution (Fig. 2D).
Substrate utilization patterns were not significantly affected
by dilution; substrates utilized in the more diluted samples
182 W.F.M. Röling et al.

Fig. 2. Effect of incubation parameters on the anaerobic Biolog GN assay. Plates were inoculated with suspensions of sediment obtained
from the border of Coupépolder landfill and underlying soil (sample T; Table 1). (A) Effect of temperature (1:100 dilution (1 g in 100 ml))
on changes in number of positive wells; 䉱, 10°C; ❍, 20–22°C; 䊏, 37°C. (B) Changes in number of positive wells for duplicate plates (1:100
dilution); 䊏, replicate 1; ❍, replicate 2. (C) Changes in percentage similarity between duplicate samples, calculated as; 䊐, simple matching
coefficient; 䉱, Jaccard coefficient. (D) Effect of dilution on development of number of positive wells; 䊏, 1:100 dilution; ❍, 1:1,000; 䉬,
1:10,000.

were in general also metabolized in the plate inoculated with
1:100 diluted sample. The 1:1000 and 1:10000 dilutions uti-
lized 4 and 3 substrates, respectively, that were not utilized in
the 1:100 diluted sample. Thus, 1:10 dilution for groundwa-
Fig. 3. Effect of gas atmosphere on percentage similarity in physi-
ological profiles, by UPGMA clustering after simple matching.
Plates were incubated for 28 days at 10°C.
ter and 1:100 dilution for sediment were used throughout
this study.
In principle, reactions in Biolog plates can also be due to
nongrowing but metabolically active bacteria. This was
tested by adding the bacteriostatic antibiotics rifampicin or
chloramphenicol to a culture of E. coli cells, exponentially
growing on glucose, and inoculating dilutions into Biolog
plates. Within a period of 3 weeks, coloring of wells was
observed only when at least 106 active cells per well were
present, also for the glucose containing well. No activity was
observed at lower dilutions, whereas plating revealed that
cells in these wells still remained culturable. Therefore, it can
be expected that the physiological profile of communities in
aquifers beneath landfills will be due to growth of the cul-
Community Profiling of Polluted Aquifers 183

Table 2. Sedimentological characterization of sediments obtained from the channel deposits and the aquifer beneath the Coupépolder
landfill

Position of core: Upstream 4 m below-landfill (channel deposits) 30 m downstr. 320 m downstr.

Codea UP-sed A-sed1 A-sed3 A-sedmix B-sed C-sed

Clay (% dw)b 1.5 2.1 5.4 2.7 2.3 1.7

Silt (% dw) 1.8 3.5 8.6 4.0 6.2 3.9
Sand (% dw) 96.7 94.4 86.0 93.3 91.5 94.4
a
See Fig. 1, Table 1.
b
% dw, percentage weight to total dry weight (dw)

turable fraction of the microbial community present, since
inoculated number of cells per well will be far less than
would result in inoculation of 106 cells.
The requirement for anaerobic incubation conditions for
obtaining a physiological profile of an anaerobic community
was tested in an experiment in which duplicate plates were
incubated under aerobic and anaerobic conditions. Highest
numbers of positive reacting substrates were observed for
the anaerobically incubated plates (73 respectively 74, with
72 substrates in common). The aerobically incubated plates
had 63 respectively 65 positive wells, with 62 substrates uti-
lized in both plates. Seven substrates were not metabolized
and thus not included for cluster analysis, while 24 sub-
strates were differentially utilized under the two conditions.
The differential utilization is also clearly expressed in the
cluster analysis (Fig. 3). Similarity between duplicates was
93% or higher but the anaerobic and aerobic incubated
plates clustered only at 70%. As a consequence, maintenance
of anaerobic conditions is essential for obtaining a relevant
physiological profile of anaerobic microbial communities.
The aquifer was anaerobic; concentrations of oxygen and
nitrate in the groundwater were below the detection limit
(<0.1 mg L-1) at the four sampling locations. Sediment
samples from the upstream reference location (UP) and
downstream locations (B and C) were sands (Table 2) and
were homogeneous in appearance. The sample from the
channel deposits beneath the landfill (location A) was very
heterogeneous: sandy, but with many thin intercalations of
organic matter and clay (Fig. 1). The channel deposits were
obviously polluted by landfill leachate, as evidenced by the
high electrical conductivity (EC) and high concentrations of
chloride, dissolved organic carbon (DOC), and BTEX (ben-
zene, toluene, ethylbenzene, xylene) in groundwater, in
comparison to the upstream reference location (Table 3). No
chlorinated compounds were detected. Values for EC, DOC,
and chloride of the two downstream locations were slightly
enhanced, but BTEX compounds were below the detection
limit (<0.8 µg L-1).
The development of the number of positive wells in the
Biolog GN plates showed that, for both groundwater and

Table 3. Hydrochemistry of groundwater collected from the

Anaerobic CLPP and DGGE Analysis of Samples from the
aquifer near the Coupépolder landfill.
Aquifer near Coupépolder Landfill
A,
The anaerobic CLPP profiling method as well as DGGE pro- UP, under B, C,
Locationa upstream landfill downstream downstream
filing were applied on groundwater and sediment samples
from the aquifer upstream, beneath, and downstream of EC (mS cm-1)b 1070 4570 1590 1220
Cl- (mg/L) 120 380 150 100
Coupépolder landfill. For sampling positions, depths and
pH 7.2 6.7 6.9 6.9
codes, see Table 1. From each sediment core at least two Alkalinity
subsamples were taken and analyzed; these subsamples are (mmol/L-1) 5.2 39 11 11
indicated by numbers. The upstream sediment sample was DOC (mg/L-1)c 13 44 20 16
BTEXd (µg/L-1) <0.8 6.0 <0.8 <0.8
only used for sedimentological analysis, as the sediment core
a
could only be sampled after 14 days because of technical See Table 1
b
EC: electrical conductivity
problems. Long-term storage is known to induce changes in c
DOC: dissolved organic carbon
microbial communities [6, 14]. d
BTEX: benzene, toluene, ethylbenzene, xylene
184
sediment, the highest number of utilized substrates was ob-
served for the polluted channel deposits underneath the
landfill (Fig. 4). This was apparent after only 7 days of in-
cubation. Of the 95 substrates present in the Biolog GN
plates, 84 were utilized in at least one case for the sediment
samples and 39 for the groundwater samples. Only the uti-
lized substrates were used for cluster analysis. Figure 5A
clearly shows that sediment physiological profiles of the
channel deposits underneath the landfill (location A) clus-
tered separately from downstream profiles, with the excep-
tion of sediment sample A-sed1. The downstream profiles
were very similar. Physiological profiles from groundwater,
including the upstream sample, had a comparable clustering;
upstream and downstream samples grouped together but
were distinct from the channel deposits (Fig. 5B). Sub-
samples of the core from the heterogeneous channel deposits
underneath the landfill contained physiologically distinct
communities, as reflected by the differences in substrates
utilized (Fig. 4) and the distant clustering (Fig. 5A). The
distinct communities might relate to the heterogeneous
composition of the core (Fig. 1). After only 1 week of incu-
bation, the clustering as shown in Fig. 5 for plates incubated
14–28 days was apparent (data not shown), despite the fact
that the number of substrates that were utilized still in-
creased (Fig. 4).
Figure 6A shows the DGGE analysis of PCR amplified 16S
rDNA fragments. Although visually already clear differences
between samples can be seen, gels were subjected to com-
puter-assisted pattern analysis using two correlation coeffi-
cients in order to determine the information content of the
W.F.M. Röling et al.
DGGE profiles. For the use of the Jaccard coefficient band-
ing positions were assigned to the gel tracks and compared.
This coefficient gives equal weight to all bands as it does not
take into account band intensity. It is sensitive to the exact
positioning of the bands. Pearson product-moment coeffi-
cient compares the densitometric curves of the tracks as a
whole. It does not require the assignment of bands. Using
Pearson product-moment correlation, very good clustering
of marker lanes from the two analyzed gels was observed; all
marker lanes grouped at 96% or higher. For correlation
using the Jaccard coefficient 17 to 28 bands were assigned
per track.
DGGE profiles of the communities in groundwater (Fig.
6A; lanes with name extension “-gw”) and sediment (lanes
with name extension “-sed”) were clearly different and clus-
tered separately with both correlation methods (Figs. 6B,C).
For the groundwater samples two groups were observed. The
upstream and the downstream communities (Fig. 6A; lanes
UP-gw, B-gw, and C-gw) were similar, but the polluted
groundwater from underneath the landfill harbored a very
different community (lane A-gw). Band-based cluster analy-
sis revealed a larger variation between the up- and down-
stream groundwater communities than clustering of the ma-
trix of Pearson product-moment coefficients. Between the
locations from which the sediment cores were obtained clear
differences in DGGE profiles were observed. However, pro-
files of the subsamples taken from each of these cores
showed that microbial communities at each sampling loca-
tion were relatively similar. This was especially true for lo-
cation B, 30 m downstream of the landfill (lane B-sed1 and
Fig. 4. Changes in number of positive wells for
groundwater and sediment samples from four lo-
cations in the aquifer near Coupépolder landfill:
䉱, channel deposits beneath landfill (location A);
❑, 30 m downstream (location B); ❍, 300 m
downstream (location C); 䉬, upstream (location
UP). Open and filled symbols were used for sedi-
ment subsamples 1 and 2, respectively. Channel
deposits sediment subsample 3 and mixture are
indicated with open and filled upside-down tri-
angles (䉲), respectively.
Community Profiling of Polluted Aquifers 185

Fig. 5. UPGMA clustering after simple matching of anaerobic

Biolog GN plates inoculated with Coupépolder samples. Similari-
ties are expressed as percentages. (A) Sediment subsamples (28 days
incubation). (B) Groundwater samples (14 days incubation). In the
sample names, capitals refer to the position from which samples
were taken (see Table 1, Fig. 1), “gw” to groundwater samples,
“sed” to sediment subsamples, numbers to sediment layers, and
“Mix” to mixture of core A.

B-sed2). The sediment subsamples (lane A-sed1, A-sed2, and

A-sed3) and mixture (lane A-sedMix) of the heterogeneous
core from the channel deposits (location A) also clustered
together, with the exception of A-sed2, when using Pearson
product-moment coefficient. This seems mainly due to dif-
ferences in intensity of individual bands in the profiles, as
agreement in banding positions clustered the samples at
higher similarity (Fig. 6B). Relatively low similarity was ob-
served between the two subsamples from location C, 300 m Fig. 6. DGGE profiles (A) and UPGMA clustering of DGGE pro-
downstream of the landfill, especially when using Pearson files (B, C) of microbial communities in sediment and groundwater
samples at Coupépolder landfill. Clustering using band-based Jac-
product-moment coefficient. Although showing low similar-
card coefficient (B) or Pearson product-moment coefficient (C)
ity, these profiles still clustered more closely to each other was expressed as percentage similarity. In the sample names, capi-
than to other profiles when band-based correlation was per- tals refer to the position from which samples were taken (see Table
formed. The separate clustering based on Pearson product- 1, Fig. 1), “gw” to groundwater sample, “sed” to sediment sub-
moment coefficient is mainly due to differences in band sample, numbers to sediment layers, and “Mix” to mixture of core
intensities between the two sub-samples (Fig. 6A; lane C- A. The markers in (A) were used to normalize gels for computer
analysis.
sed1 and C-sed2).

anaerobic conditions was developed. This method was used

Discussion
to characterize the microbial community in an anaerobic
A new standardized method for the determination of aquifer beneath a landfill, but can also be applied for samples
community-level physiological profiles (CLPP) under from other anaerobic environments such as human or
186
animal intestines and anaerobic wastewater treatment
plants.
For analysis of the physiological profile of anaerobic com-
munities at landfill leachate polluted aquifers, incubation at
the in situ temperature of the aquifer (10°C) during 28 day
incubation was optimal for obtaining a reproducible anaero-
bic physiological profile (see Fig. 2). Samples should be di-
luted as little as possible since the number of microorgan-
isms usually will be below 107/ml or g [4, 16, 20], of which
only a minor part likely will be culturable [1]. Growth in the
wells of the plate is essential for reduction of the tetrazolium
dye. Furthermore, a specific profile of anaerobic communi-
ties can only be obtained by using anaerobic incubation
conditions.
Although in this study scoring negative and positive wells
enabled the separation of treatments (Fig. 3) or samples (Fig.
5), it can be expected that quantitative measurements using
a microplate reader will yield even more informative data
and will further improve separation of samples. The use of
Biolog MT plates, filled with specific substrates such as car-
bon sources found in landfill leachate and degradation in-
termediates, will aid in obtaining a physiological profile that
is more relevant for landfill leachate polluted aquifers. This
approach has been used to determine the biodegradation
potential in wastewater treatment systems [26].
The anaerobic physiological profiling method was devel-
oped as part of our effort to establish a database linking,
among others, composition of microbial communities to
intrinsic bioremediation. Another part of the research aimed
at testing this method in combination with denaturing gra-
dient gel electrophoresis (DGGE) for their suitability as
simple profiling methods, which yield large amounts of in-
formation for inclusion into such a database. The methods
were applied to groundwater and sediment obtained up-
stream, beneath, and downstream of the Coupépolder land-
fill, Alphen aan de Rijn, The Netherlands. At all locations
anaerobic conditions were encountered, including the pris-
tine upstream location. In The Netherlands many pristine
aquifers are anaerobic because of oxygen consumption or
limitation of oxygen diffusion in organic and impermeable
clayey soils respectively. The location underneath the landfill
was clearly polluted by landfill leachate, but this was less
obvious for the downstream locations. Both CLPP (Fig. 5)
and DGGE (Fig. 6) showed their suitability for distinguish-
ing microbial communities in groundwater and sediment at
the polluted location from the microbial communities at the
pristine upstream location and less-polluted downstream lo-
cations. The higher number of positive reacting wells ob-
W.F.M. Röling et al.
served for both sediment and groundwater underneath the
landfill (Fig. 4) might be related to the inflow of carbon-rich
landfill leachate into the aquifer. Dissolved organic carbon
(DOC) was 44 mg L-1 and higher while at downstream lo-
cations the concentration was only about 15 mg L-1 (Table
3). The leachate is likely to contain more easily degradable
carbon sources, supporting a more physiologically diverse
community.
A combination of cultivation-dependent CLPP and cul-
tivation-independent 16S rDNA based DGGE profiling of
both groundwater and sediment samples yielded the best
separating power for characterizing microbial communities
in aquifers. Differentiation between the microbial commu-
nities at the upstream and downstream locations was only
observed via DGGE analysis of the sediment samples, which
showed a location-specific DGGE profile. These locations
could not be separated via CLPP analysis. Alternatively,
CLPP analysis differentiated sediment subsample A-sed1
from the channel deposits underneath the landfill from A-
sed3 and A-sedMix, which all had similar DGGE profiles.
Subsample A-sed2 was similar in CLPP to A-sed3 and A-
sedMix, but showed a different DGGE profile. DGGE analy-
sis also revealed obvious differences between microbial com-
munities in corresponding groundwater and sediment
samples (Fig. 6), showing that the much more cost-effective
and easier sampling of groundwater is not sufficient to ob-
tain a relevant picture about microbial communities in the
aquifer near Coupépolder landfill.
The two correlation methods used for computer-assisted
DGGE pattern analysis, the band-based Jaccard coefficient
and the whole-densitometric-curve-considering Pearson
product-moment coefficient, were also complementary.
Profiles with similar banding patterns sometimes clustered
more distinctly when the quantitative information of the
densitometric curve was taken into account, as the case for
the sediment samples from location A (Fig. 6). Clustering
based on the whole densitometric curve sometimes failed to
reveal differences in DGGE patterns because of the presence
of minor bands, as obvious for the up- and downstream
groundwater samples.
Microbial communities within the aquifer varied both on
a large spatial scale, as shown by the different grouping of
DGGE profiles of sediment from the different locations, and
on a small spatial scale, as shown by CLPP and DGGE analy-
sis on the core from the channel deposits. This heterogeneity
should be taken into account in a sampling program for
setting up a database, as it might enhance intrinsic biore-
mediation. One can imagine that when landfill leachate
Community Profiling of Polluted Aquifers
moves through heterogeneous microbial environments,
there will be a higher chance that the leachate will encounter
microorganisms having the capacities to degrade the pollut-
ants it contains. Although the microbial communities at the
polluted location differed from those at the un- or less-
polluted locations, we are as yet unable to link (members of)
microbial communities to bioremediation potential. Infor-
mation derived from hydrogeochemical properties and deg-
radation tests is required for establishing this link. Such
analyses should be included in the sampling program for
establishing a database.
Acknowledgments
This investigation was financially supported by the Dutch
Research Program Biotechnological In-situ Remediation
(NOBIS) and the province of Zuid-Holland, The Nether-
lands.
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