Geological Survey of Denmark and Greenland Bulletin
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Nr. 4, Review of Survey activities 2003, pp. 33-36 |
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33
The first DNA-based methods for direct quantification of
soil protozoa, and a DNA-based quantification method to
describe the spread of phenanthrene-degrading bacteria in
soil and freshwater aquifers, have recently been developed at
the BIOPRO Research Centre at the Geological Survey of
Denmark and Greenland (GEUS). Well-known genes for
phenoxyalcanoic acid degradation have been used to monitor
the in situ degradation of phenoxyalcanoic acid pesticides.
Studies have been initiated on the short-lived mRNA mole-
cules that are expected to provide a shortcut to the under-
standing of low, yet important, microbial activity in
geological samples. This article reviews recent developments
soil protozoa, and a DNA-based quantification method to
describe the spread of phenanthrene-degrading bacteria in
soil and freshwater aquifers, have recently been developed at
the BIOPRO Research Centre at the Geological Survey of
Denmark and Greenland (GEUS). Well-known genes for
phenoxyalcanoic acid degradation have been used to monitor
the in situ degradation of phenoxyalcanoic acid pesticides.
Studies have been initiated on the short-lived mRNA mole-
cules that are expected to provide a shortcut to the under-
standing of low, yet important, microbial activity in
geological samples. This article reviews recent developments
in techniques based on analysis of nucleic acids from soils and
aquifers.
aquifers.
Analytical work has been carried out mainly on soil sam-
ples from a former asphalt production plant at Ringe (Fig. 1).
The Ringe plant constitutes one of the most polluted indus-
trial sites in Denmark, and is a priority site of studies by the
BIOPRO Research Centre. Although rich in carbon, the
Ringe subsoil is an oligotrophic environment due to the high
content of polycyclic aromatic hydrocarbons (PAH). This is
an environment where the supply of nutrients to microor-
ganisms is low, leading to slow growth, low total numbers of
microorganisms and small cells. To study microbial commu-
nities of oligotrophic environments, analytical methods with
low detection limits are needed. Until recently, microorgan-
isms of natural environments were mainly studied by cultiva-
tion-dependent methods. However, microorganisms that can
be cultured on agar plates are now known to represent only a
small fraction of the total microbial community. Modern
methods, therefore, need to be based on the detection of bio-
molecules in the microorganisms rather than being depen-
dent on growth of the microorganisms. The best available
techniques are based on DNA and RNA molecules (Fig. 2),
The Ringe plant constitutes one of the most polluted indus-
trial sites in Denmark, and is a priority site of studies by the
BIOPRO Research Centre. Although rich in carbon, the
Ringe subsoil is an oligotrophic environment due to the high
content of polycyclic aromatic hydrocarbons (PAH). This is
an environment where the supply of nutrients to microor-
ganisms is low, leading to slow growth, low total numbers of
microorganisms and small cells. To study microbial commu-
nities of oligotrophic environments, analytical methods with
low detection limits are needed. Until recently, microorgan-
isms of natural environments were mainly studied by cultiva-
tion-dependent methods. However, microorganisms that can
be cultured on agar plates are now known to represent only a
small fraction of the total microbial community. Modern
methods, therefore, need to be based on the detection of bio-
molecules in the microorganisms rather than being depen-
dent on growth of the microorganisms. The best available
techniques are based on DNA and RNA molecules (Fig. 2),
which due to their high level of resolution
allow closely related organisms or func-
tional genes to be distinguished. In the fol-
lowing review, examples are given of appli-
cations of these nucleic acid based methods.
allow closely related organisms or func-
tional genes to be distinguished. In the fol-
lowing review, examples are given of appli-
cations of these nucleic acid based methods.
Direct analysis of microbial populations in soil and fresh-
water aquifers using nucleic acid based techniques
water aquifers using nucleic acid based techniques
Carsten S. Jacobsen, Julia R. de Lipthay, Mikkel Bender, Line Fredslund, Anders R. Johnsen and
Kaare Johnsen
Kaare Johnsen
Fig. 1. Location of the former asphalt plant at Ringe, one of the most polluted
industrial sites in Denmark. The BIOPRO Research Centre at GEUS monitored in
situ degradation of phenoxyalcanoic acid pesticides. The pits, sampling sites and
wells were used to determine the distribution of contamination of tar (pale red)
and kerosene (green) in the subsoil. A field injection experiment, using a mixture
of six different herbicides was carried out at a shallow sandy aquifer at Vejen.
Modified from Rosenbom et al. (2000).
Geological Survey of Denmark and Greenland Bulletin 4, 3336 (2004) © GEUS, 2004
DNA analysis is not limited to living
organisms
organisms
Unlike other microbial analysis tools, DNA analyses may be
carried out on both living and dead material, as long as the
DNA has not been degraded. For analysis of DNA in soils, a
major problem is the presence of humic substances, that even
at low concentrations interfere with the enzymes used in the
DNA amplification process. All research groups working
with soil nucleic acids experience problems with amplifica-
tion of DNA when soils contain much humic material. One
way to solve this problem is to use selective purification
methods in which single-stranded DNA is selectively puri-
fied from the soil using a molecular `fishing rod' equipped
with the complementary DNA strand.
carried out on both living and dead material, as long as the
DNA has not been degraded. For analysis of DNA in soils, a
major problem is the presence of humic substances, that even
at low concentrations interfere with the enzymes used in the
DNA amplification process. All research groups working
with soil nucleic acids experience problems with amplifica-
tion of DNA when soils contain much humic material. One
way to solve this problem is to use selective purification
methods in which single-stranded DNA is selectively puri-
fied from the soil using a molecular `fishing rod' equipped
with the complementary DNA strand.
After extracting the gene of interest, it is possible to mul-
tiply its numbers using the `Polymerase Chain Reaction'
(PCR; Saiki et al. 1985). PCR is an exponential reaction in
which a single DNA strand can, in principle, produce four
million identical copies by 25 cycles of multiplication. In
PCR, two small DNA sequences, corresponding to two
regions on the gene, are selected as priming sites for two seg-
ments of complementary DNA. These small DNA pieces are
designated `primers'. The primers serve as the target of the
PCR, and lead to the formation of a large number of DNA
molecules identical to the original gene. The primers are con-
structed by consulting DNA sequence databases on the
Internet. Since these databases are very comprehensive, it is
possible to construct primer sets that are specific for the
desired taxonomic or functional groups.
(PCR; Saiki et al. 1985). PCR is an exponential reaction in
which a single DNA strand can, in principle, produce four
million identical copies by 25 cycles of multiplication. In
PCR, two small DNA sequences, corresponding to two
regions on the gene, are selected as priming sites for two seg-
ments of complementary DNA. These small DNA pieces are
designated `primers'. The primers serve as the target of the
PCR, and lead to the formation of a large number of DNA
molecules identical to the original gene. The primers are con-
structed by consulting DNA sequence databases on the
Internet. Since these databases are very comprehensive, it is
possible to construct primer sets that are specific for the
desired taxonomic or functional groups.
This technique has, for example, enabled forensic experts
to produce enough DNA molecules to determine whether
the genomic fingerprint of a person matches that of a blood-
stain at the scene of a crime. We have used this technique
widely for the analysis of microorganisms in the environ-
ment, and the detection limit is less than 40 cells in a sample
(Jacobsen 1995). The challenge is now not only to detect, but
also to quantify the DNA from very few cells in soil and
freshwater aquifers.
the genomic fingerprint of a person matches that of a blood-
stain at the scene of a crime. We have used this technique
widely for the analysis of microorganisms in the environ-
ment, and the detection limit is less than 40 cells in a sample
(Jacobsen 1995). The challenge is now not only to detect, but
also to quantify the DNA from very few cells in soil and
freshwater aquifers.
Quantitative DNA techniques for the
enumeration of soil flagellates and bacteria
enumeration of soil flagellates and bacteria
As a consequence of the exponential nature of PCR, it is an
excellent technique for the detection of specific DNA
sequences. On the other hand, it is not quantitative, and
small differences in the efficiency of the reaction affect the
final number of DNA copies. Several methods have been
proposed to resolve this problem. One possibility is using the
principle of `most probable number' (MPN) estimates, where
the DNA template is serially diluted in several replicate reac-
tions. By looking at which dilutions of template DNA that
give a product from the PCR, and in how many of the repli-
cate reactions, an estimate of the original number of genes in
the sample can be made. This approach has been used to
develop the first successful molecular detection and quantifi-
cation of protozoa in soil (Fredslund et al. 2001). This new
technique represents a breakthrough in reliable enumeration
of soil protozoa, since these often small and amoeboid organ-
isms are difficult to enumerate using microscopy techniques.
Traditionally, the enumeration was based on growth-depen-
dent techniques, where the cultivable fraction of the total
protozoan populations was not known. In our study, a part of
the 18S rDNA of the common soil flagellate Heteromita glo-
bosa was sequenced and PCR primers for this gene were
developed. In a sterilised soil at the Ringe asphalt production
plant, the population dynamics of this flagellate and the
phenanthrene-degrading bacterium Pseudomonas putida
OUS82 were quantified using both growth-dependent tech-
niques and the MPN-PCR assay (Fredslund et al. 2001).
excellent technique for the detection of specific DNA
sequences. On the other hand, it is not quantitative, and
small differences in the efficiency of the reaction affect the
final number of DNA copies. Several methods have been
proposed to resolve this problem. One possibility is using the
principle of `most probable number' (MPN) estimates, where
the DNA template is serially diluted in several replicate reac-
tions. By looking at which dilutions of template DNA that
give a product from the PCR, and in how many of the repli-
cate reactions, an estimate of the original number of genes in
the sample can be made. This approach has been used to
develop the first successful molecular detection and quantifi-
cation of protozoa in soil (Fredslund et al. 2001). This new
technique represents a breakthrough in reliable enumeration
of soil protozoa, since these often small and amoeboid organ-
isms are difficult to enumerate using microscopy techniques.
Traditionally, the enumeration was based on growth-depen-
dent techniques, where the cultivable fraction of the total
protozoan populations was not known. In our study, a part of
the 18S rDNA of the common soil flagellate Heteromita glo-
bosa was sequenced and PCR primers for this gene were
developed. In a sterilised soil at the Ringe asphalt production
plant, the population dynamics of this flagellate and the
phenanthrene-degrading bacterium Pseudomonas putida
OUS82 were quantified using both growth-dependent tech-
niques and the MPN-PCR assay (Fredslund et al. 2001).
Alternative methods for quantification of DNA are the
real-time PCR and the competitive PCR methods. In real-
time PCR, the DNA multiplication is monitored on-line
using a combination of fluorescent DNA stains and fibre
optics coupled to a computer. GEUS has recently received
funding from the Danish Natural Science Research Council
to implement this technique.
time PCR, the DNA multiplication is monitored on-line
using a combination of fluorescent DNA stains and fibre
optics coupled to a computer. GEUS has recently received
funding from the Danish Natural Science Research Council
to implement this technique.
Competitive PCR makes use of an internal standard in the
form of a similar, but shorter DNA molecule, which is recog-
nised and hence amplified by the same primer set as the tem-
plate DNA (the DNA that needs to be quantified). The
nised and hence amplified by the same primer set as the tem-
plate DNA (the DNA that needs to be quantified). The
34
Fig. 2. All information regarding cell function and structure is contained
as a genetic code in the cell DNA. Each gene encodes a specific function
by dictating the synthesis of a specific protein. Before proteins are syn-
thesised, the genes are copied (transcribed) into messenger RNA
(mRNA). After transcription, the mRNA is translated into protein by pro-
tein-synthesising machinery called ribosomes. The sequence of bases in
the mRNA, copied after the base sequence in the gene, determines the
structure and function of the protein.
internal standard is added to the reaction mixtures in decreas-
ing amounts and competes with the template DNA for
amplification. Thus, the amount of product from the inter-
nal standard is inversely related to the initial amount of the
template DNA. Competitive PCR exploits the highly sensi-
tive nature of the PCR process, while using an internal stan-
dard to bypass the quantification problems inherent in the
amplification reaction (Johnsen et al. 1999).
ing amounts and competes with the template DNA for
amplification. Thus, the amount of product from the inter-
nal standard is inversely related to the initial amount of the
template DNA. Competitive PCR exploits the highly sensi-
tive nature of the PCR process, while using an internal stan-
dard to bypass the quantification problems inherent in the
amplification reaction (Johnsen et al. 1999).
Microbial changes in aquifers contaminated
with phenoxyalcanoic acid herbicides
with phenoxyalcanoic acid herbicides
This section focuses on specific genes of interest rather than
on organisms. Phenoxyalcanoic acid herbicides are exten-
sively used in agriculture, and include compounds such as
mecoprop (MCPP), 2,4-dichlorophenoxyacetic acid (2,4-D)
and dichlorprop. A common pathway for 2,4-D degradation
has been determined for the bacterial strain Ralstonia
eutropha JMP134, and the catabolic genes (tfd) encoding the
specific enzymes have been identified (Fig. 3; Don et al.
1985). The in situ adaptation processes of the indigenous
microorganisms when exposed to these herbicides have been
investigated by studying the impact on a microbial commu-
nity in a freshwater aquifer. A field injection experiment was
carried out at a shallow sandy aquifer at Vejen, Denmark (Fig.
1). During a seven-month period, a mixture of six different
on organisms. Phenoxyalcanoic acid herbicides are exten-
sively used in agriculture, and include compounds such as
mecoprop (MCPP), 2,4-dichlorophenoxyacetic acid (2,4-D)
and dichlorprop. A common pathway for 2,4-D degradation
has been determined for the bacterial strain Ralstonia
eutropha JMP134, and the catabolic genes (tfd) encoding the
specific enzymes have been identified (Fig. 3; Don et al.
1985). The in situ adaptation processes of the indigenous
microorganisms when exposed to these herbicides have been
investigated by studying the impact on a microbial commu-
nity in a freshwater aquifer. A field injection experiment was
carried out at a shallow sandy aquifer at Vejen, Denmark (Fig.
1). During a seven-month period, a mixture of six different
herbicides, including MCPP and dichlorprop, was continu-
ously injected into the aquifer creating a contaminant plume
(Broholm et al. 2000).
ously injected into the aquifer creating a contaminant plume
(Broholm et al. 2000).
Sediment and groundwater samples from herbicide-
exposed (1 and 2) and non-exposed (NX) sites (Fig. 4A) were
collected, and the impact on microbial community structure
and function was studied (de Lipthay et al. 2000). Laboratory
incubations demonstrated that sediment samples collected
inside the contaminant plume had acquired a significantly
increased capacity for herbicide mineralisation compared to
samples from non-exposed sites (Fig. 4A). Thus, the in situ
exposure to herbicides resulted in microbial communities
that were better adapted to the degradation of phenoxyal-
canoic acids. This was further demonstrated by greatly
increased populations of pesticide degraders inside the pesti-
cide plume, both when enumerated by cultivation, and when
quantified by the number of pesticide genes (tfdABC)
detected by PCR methods (Fig. 4B, treatment 1 and 2).
Pesticide degraders and their tfd genes were undetectable out-
side the plume (Fig. 4B, treatment NX). The most likely
collected, and the impact on microbial community structure
and function was studied (de Lipthay et al. 2000). Laboratory
incubations demonstrated that sediment samples collected
inside the contaminant plume had acquired a significantly
increased capacity for herbicide mineralisation compared to
samples from non-exposed sites (Fig. 4A). Thus, the in situ
exposure to herbicides resulted in microbial communities
that were better adapted to the degradation of phenoxyal-
canoic acids. This was further demonstrated by greatly
increased populations of pesticide degraders inside the pesti-
cide plume, both when enumerated by cultivation, and when
quantified by the number of pesticide genes (tfdABC)
detected by PCR methods (Fig. 4B, treatment 1 and 2).
Pesticide degraders and their tfd genes were undetectable out-
side the plume (Fig. 4B, treatment NX). The most likely
35
Fig. 3. Pathway for degradation of 2,4-D as elucidated in the bacterial
strain Ralstonia eutropha JMP134. The tfdA gene encodes a 2,4-D dioxy-
genase, tfdB encodes a 2,4-dichlorophenol hydroxylase, and tfdC en-
codes a chlorocatechol 1,2-dioxygenase. The 2,4-dichloromuconic acid
generated by the activity of the tfdC gene product is further transformed
to intermediates of the tricarboxylic acid cycle by the activity of other tfd
gene products.
Fig. 4. A: Mineralisation of the phenoxyalcanoic acid herbicides 2,4-D
(red) and MCPP (blue) in laboratory incubations of sediment samples
from herbicide exposed (1, 2) and non-exposed (NX) sites of the Vejen
aquifer. B: Effect of in situ herbicide exposure in two exposed (1, 2) sites
on microbial biomass of 2,4-D and MCPP degraders, and on the presence
of tfdA, tfdB and tfdC genes. Data show that indigenous microbial com-
munities carry the tfd genes and are capable of degrading phenoxyal-
canoic acids.
explanation is that microorganisms carrying the tfd genes had
a selective advantage in that they could make use of the pes-
ticides as sources of carbon and energy. The data suggest that
natural attenuation is a likely procedure for clean up of this
group of herbicide compounds when originating from point-
source contaminations.
a selective advantage in that they could make use of the pes-
ticides as sources of carbon and energy. The data suggest that
natural attenuation is a likely procedure for clean up of this
group of herbicide compounds when originating from point-
source contaminations.
Analysis of microbial activity applying
mRNA techniques
mRNA techniques
The presence or absence of specific microorganisms may be
determined by use of cultivation or DNA-based techniques,
although these methods give no information as to whether
the organisms are actually active in the environment.
Microbial activity may be measured in several ways. The first
sign of activity in microbial cells is the synthesis of messen-
ger-RNA (mRNA; Fig. 2). These molecules have half-lives of
only a few minutes, and the detection of mRNA thus ensures
that the genes of interest are actually expressed at the time of
sampling. Another approach is to detect the activity of the
gene products the enzymes. However, the longevity of
enzyme activities is variable. A third approach is to measure
the target molecules of the enzymes, i.e. the pollutant mole-
cules. By use of analytical chemical methods such as gas and
liquid chromatography, the concentration of target mole-
cules may be measured, and the dissipation of pollutants
indicates microbial activity.
determined by use of cultivation or DNA-based techniques,
although these methods give no information as to whether
the organisms are actually active in the environment.
Microbial activity may be measured in several ways. The first
sign of activity in microbial cells is the synthesis of messen-
ger-RNA (mRNA; Fig. 2). These molecules have half-lives of
only a few minutes, and the detection of mRNA thus ensures
that the genes of interest are actually expressed at the time of
sampling. Another approach is to detect the activity of the
gene products the enzymes. However, the longevity of
enzyme activities is variable. A third approach is to measure
the target molecules of the enzymes, i.e. the pollutant mole-
cules. By use of analytical chemical methods such as gas and
liquid chromatography, the concentration of target mole-
cules may be measured, and the dissipation of pollutants
indicates microbial activity.
A major topic of future studies in microbial ecology will
be the assessment of microbial activity by the application of
mRNA techniques to answer which microorganisms are
active, and under which conditions their genes are expressed.
mRNA techniques to answer which microorganisms are
active, and under which conditions their genes are expressed.
The two most commonly used techniques for detection of
mRNA (Fig. 2) are reverse transcription polymerase chain
reaction (RT-PCR) and RNA-RNA hybridisation.
reaction (RT-PCR) and RNA-RNA hybridisation.
In RT-PCR, the first step is a reverse transcription process
the conversion of mRNA into copy DNA (cDNA). Reverse
transcription requires a small DNA primer to bind to the
mRNA in order to initiate synthesis of cDNA. Thus, specific
mRNAs can be amplified by using sequence-specific primers
in the RT-PCR reaction. Following synthesis of cDNA, a
normal PCR is carried out to multiply the cDNA, and the
resulting PCR products are detected by usual DNA detection
techniques.
transcription requires a small DNA primer to bind to the
mRNA in order to initiate synthesis of cDNA. Thus, specific
mRNAs can be amplified by using sequence-specific primers
in the RT-PCR reaction. Following synthesis of cDNA, a
normal PCR is carried out to multiply the cDNA, and the
resulting PCR products are detected by usual DNA detection
techniques.
Direct RNA-RNA hybridisation analyses exploit a com-
pletely different principle; this is directly quantitative but
lacks the sensitivity of RT-PCR. First, the total content of
mRNA is extracted. Then the mRNA of interest is identified
by binding of a specific RNA probe with a sequence comple-
lacks the sensitivity of RT-PCR. First, the total content of
mRNA is extracted. Then the mRNA of interest is identified
by binding of a specific RNA probe with a sequence comple-
mentary to the mRNA gene of interest (hybridisation). By
using a `radiolabelled' probe, the final quantification of the
mRNA of interest is easily done by determining the amount
of `radiolabelled' bound to the mRNA.
using a `radiolabelled' probe, the final quantification of the
mRNA of interest is easily done by determining the amount
of `radiolabelled' bound to the mRNA.
A study using freshwater samples artificially contaminated
with the herbicide 2,4-D, revealed a significant increase in
the amount of tfdA mRNA, as measured by hybridisation of
RNA extracts using a tfdA specific probe. This demonstrates
that the 2,4-D degraders in the freshwater samples were
actively degrading the 2,4-D (Fig. 5). Transcription of tfdA
was, however, transient and the degradation of 2,4-D con-
tinued although mRNA levels dropped. These observations
illustrate that the herbicide-degrading enzymes encoded by the
mRNA last longer in the cells than the mRNA `signal' itself.
the amount of tfdA mRNA, as measured by hybridisation of
RNA extracts using a tfdA specific probe. This demonstrates
that the 2,4-D degraders in the freshwater samples were
actively degrading the 2,4-D (Fig. 5). Transcription of tfdA
was, however, transient and the degradation of 2,4-D con-
tinued although mRNA levels dropped. These observations
illustrate that the herbicide-degrading enzymes encoded by the
mRNA last longer in the cells than the mRNA `signal' itself.
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36
Authors' address
Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. E-mail: csj@geus.dk
Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. E-mail: csj@geus.dk
