and such work is significant for a better understanding of
ecological processes in leachate polluted aquifers.
Limitations of the traditional cultivation based
techniques have restricted our knowledge of the microbiology
in various ecosystems. Recently,several studies on microbial
communities using 16S rRNA gene analysis have been
reported.They have provided unprecedentedly
detailed insights into the microbial communities.Spatial
differences among microbial communities in leachate polluted
aquifers have been described using molecular approaches.However ,to our knowledge, there has
been no comparable investigation about variations of dominant
microbial populations in groundwater subject to a landfill with
time.
This paper describes temporal variations of
hydrochemical conditions and dominant microbial populations
in response to the pollution of leachate.Dominant microbial
populations were determined using restriction fragment length
polymorphism(RFLP) analyses of 16S rRNA gene libraries.
1 　Materials and methods
1. 1 　Sampling
The Laogang Landfill was separated into many
compartments by clay dams,with 10 hectares for each
compartment.A spare compartment was scheduled to operate
at the end of 2002,accordingly,a monitor well which is 10
m downstream from the border of this compartment was
chosen for our investigation.Before this compartment was put
to operation,groundwater was sampled in November 2002;
after the compartment began operation,groundwater was
sampled twice more in January and May 2003.Samples of 3
different dates were named as A,B and C by time sequence.

Sampling procedures of groundwater :after 3 volumes of
standing water of the monitor well was removed with a special
device,groundwater was collected into 500 ml pre2sterilized
and nitrogen flushed PVC bottles,which were filled to
capacity and tightly capped with screw cap.Samples were
transferred to our laboratory on ice and stored at 4 ℃for less
than 12 h. Fresh groundwater samples were filtrated with 150
mm diameter,0122μm pore2size filters by air2compressing ,
and filters were frozen at - 80 ℃until DNA extraction.
1. 2 　Chemical analysis
At the sampling location,the temperature,pH,and
dissolved oxygen were measured in situ,and the
concentrations of H S and CH in the headspace of the
monitor well also were measured by a portable gas analyzer.
Other hydrochemical analyses were performed following
procedures described by “Chinese national measurement
standards for oceanic environments ”.
Parameters measured included total dissolved solid,total
organic carbon,SO ,S ,chemical oxygen demand,Cl ,
NH N,NO N,F ,hardness,and metals.
1. 3 　DNA extraction and 16S rRNA gene a mplification
Filters containing microbes were cut in small pieces,
and DNA was extracted by using R°ling’s method (R°ling,
2000 ).After purifying by using the 3S DNA column
purification kit (Biocolor,China),the DNA was used as a
template to amplify 16S rRNA gene using the universal
primers 27f and 1492r.PCR reaction mixtures as well as
thermal cycling were performed under the conditions
described by Martin Laurent (Martin Laurent,2001) with the
GeneAmp PCR System 2400(Applied Biosystems,USA).
1. 4 　Construction of 16S rRNA gene libraries

The PCR amplified 16S rRNA gene products were
purified by using the 3S PCR product purification kit
(Biocolor) prior to ligation.Clone libraries were constructed
using the TVector PCR product cloning kit (Biocolor ).
Clones were screened with PCR for the presence of the inserts
using primers in separate PCR reactions. If its
length were approximate 1550 bp,the PCR product of the
clone would be further analyzed by restriction fragment length
polymorphism(RFLP).
1. 5 　RFLP analyses and sequencing
The PCR products were separately digested with
restriction enzymes MspI and RsaI (Sibenzyme) for 2 h at
37 ℃.The above digests were resolved on 2 % agarose gels,
and a molecular weight marker was included in each gel.The
gel was stained with ethidium bromide and the pattern was
documented using a gel documentation system( GelDoc2000,
Bio2Rad,USA).Gel images were converted,normalized,
and analyzed with the Diversity Database 2. 2. 0 software
(Bio2Rad).
Purified PCR products of the selected clones from RFLP
analyses were used as the sequencing template,and DNA
sequences were determined with automated fluorescent Taq
cycle sequencing using the ABI 377 Sequencer (Applied
Biosystems).
2 　Results and discussion
The microbes in groundwater was investigated rather
than sediment associated microbes,due to the fact that a
large portion of the sediment bound microbes could be
physically ( e.g.,in pores ) or biologically ( e.g.,in
biofilms) protected from the influence of environmental
perturbations.Research on groundwater could generate more

specific data on how variations in environmental conditions
are reflected in changes of the dominant microbial
populations.
2. 1 　Hydrochemical changes of groundwater
The values of hydrochemical parameters of groundwater
sample A,B and C are shown in Table1.Leachate is rich in
dissolved organic matter,inorganic species and heavy metals,
which may be reflected by parameters such as COD(chemical
oxygen demand),NH N,TOC(total organic carbon),TDS
(total dissolved solid),Cl ,hardness,Pb,Ni and Cr.
There were obvious increases in values of these parameters
from A to C.These increases indicated that the leachate from
that compartment had made a impact on the downstream
groundwater.
　　Depending on the impact of the leachate and the redox
buffering capacity of aquifers,a sequence of anaerobic redox
zones (e.g.methane production,sulfate,iron,manganese,
and nitrate reduction) may develop in aquifers,and this
sequence strongly affects the behavior of the pollutants
leaching from the landfill.
Determination of the dominating redox process by
composition of dissolved redox relevant species in groundwater
has been proved to be effective (Ludvigsen,1998).After
analyses of redox relevant species (DO,NO N,Fe,Mn,
SO ,S ,H S ,CH ),the dominating redox process have
been roughly determined.It was estimated that nitrate (33. 2
mgPL) was likely to be the dominant electron acceptor and
accordingly denitrification would be a major redox process for
the groundwater represented by the sample A,because the
contents of DO,Fe,Mn were very low,and H S and CH

were not detected. Likewise,methane production and
sulfate reduction were deduced to be simultaneous major
redox processes for the groudwater represented by both B and
C,since CH ,H S were detected and the concentration of
other dissolved redox relevant ions were low.It is apparent
that landfilling has produced a profound effect on redox
conditions of the underlying aquifer.
It is noticeable that the values of TDS,Cl and
hardness are remarkably high,even before that compartment
was landfilled ( sample A).Leachate alone is not likely to
raise these values to such a high level as referred to data from
non seashore landfills.Seawater was deduced to be
responsible for this situation,because seashore aquifers
should have been inevitably infiltrated by seawater with the
flowing tide.It is worthy of further investigation since
combined pollution by leachate and seawater have turned
these aquifers into environments of special physicochemical
condition.
2. 2 　Dominant microbial populations in groundwater
samples
In the process of construction of 16S rRNA gene
Libraries,blue white selection was used for screening,and
120 white colonies were randomly picked for each sample.A
total of 113,115 and 108 clones with correct inserts were
selected for samples A,B and C.
A large variety of different RFLP patterns was found
when the clones were analyzed by MspI and RsaI digestions.
The combined genotype was defined as a group of sequences
that have indistinguishable MspI and RsaI restriction
patterns.Accordingly,there were 45,49 and 48 combined
genotypes to be grouped for sample A,B and C,

respectively.From a statistical standpoint,each combined
genotype was generally regarded as a unique population(Yu,
2001).
From 45,49 and 48 combined genotypes in A,B and C
Libraries,there were 7,8 and 7 including 4 clones or more,
which been regarded as the representatives of the dominant
microbial populations.Inserts of one clone from each
dominant combined genotype were successfully partially
sequenced (≈600 bp ).Sequences were assigned to major
microbial groups and the closest known relatives were chosen
by searching in GenBank databases based on BLAST
similarities.Summary of phylogenetic affiliation of the
dominant microbial populations are shown in Table 2.
There is strong dominance by bacteria belonging to β2
Proteobacteria in the A library,while no clones showed
affiliation to β2 Proteobacteria,and relatively dominant
microbial groups are gram positive bacteria (e. g. Firmicutes,
Fusobacteria) in the libraries B and C. The results indicated
that there is a complete shift in the composition of the
dominant populations after landfilling.On the other hand,
there is no clear transition of dominant microbial populations
found between the sample B and C.
　　Based on the GenBank’s indications of the closest
relative of a retrieved sequence , a possible biochemical role
of the bacterium represented by the sequence could be
surmised. It should be noted that 1 sequence related to
genera capable of denitrification ( Azoarcus related ) were
retrieved.Remarkably,3 sequences are closely related to
sulfate reducers.Three sequences are associated with the
methanotrophic bacteria though no sequence affiliated with
methanogenic bacterium was encountered.This situation is in

accordance with major redox processes in these environments
implied by hydrochemical analyses.It was found that several
redox processes could take place simultaneously in many
cases ( Ludvigsen,1998 ),which is consistent with
coexistence of sulfate reducing bacteria and methanotrophic
bacteria in groundwater samples B and C.
A phylogenetic descri ption of dominant microbial
populations is an important step in understanding of ecological
processes in aquifers beneath a landfill.However,
phylogenetic affiliation alone is not sufficient to firmly connect
certain microbial taxa with a biochemical reaction,therefore
we can only hypothesize about its possible biochemical role.
On the other hand,evidences from other aspects can be used
to support the validity of our speculations.It is reasonable
that more sequences are related to complex compound
degrading fermentative bacteria in the samples B and C. For
example,2 sequences (3JL288 and 3ML292) are affiliated
with aminobacterium colombiense,an aminoacid degrading
obligate anaerobe.A sequence ( 3JL293) is affiliated with
Ilyobacter polytropus,a fermentative Fusobacterium which
specialized in the degradation of hydroaromatic compounds.
In this study,it is significative that phylogenetic information
showing variations of dominant microbial populations is in
accordance with hydrochemical changes of groundwater on the
whole.
3 　Conclusions
The results indicated that obvious changes of dominant
microbial populations had occurred in groundwater in
response to leachate from Laogang Landfill.Concentration
variations of dissolved redox relevant species suggested that
the dominating redox process had changed from denitrification
to methane production Psulfate reduction. The dominating
redox process determined is accordant with the closest
relatives of some dominant clones,based on their indications
on the ability to perform redox reactions in the GenBank
database.