Soil Biology & Biochemistry 35 (2003) 1507–1516
www.elsevier.com/locate/soilbio
Changes in the succession and diversity of protozoan and microbial
populations in soil spiked with a range of copper concentrations
Flemming Ekelunda,*, Stefan Olssonb, Anders Johansenb
a
Department of Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen Ø, Denmark
Department of Environmental Chemistry and Microbiology, Danish Environmental Research Institute, Frederiksborgvei 399, DK-4000 Denmark
b
Received 17 January 2002; received in revised form 16 June 2003; accepted 1 July 2003
Abstract
We studied microbial and protozoan activity, diversity and abundance as affected by Cu2þ amendments ranging from 0 to 1000 mg g21
over a 70-day period. At the end of the experiment the microbial population size, as indicated by substrate-induced respiration, had
normalized for all Cu2þ concentrations, but 1000 mg g21. Protozoan abundance was negatively affected by Cu2þ, although, only in the first
few weeks. A more detailed analysis of the individual components that make up the microbial and micro-faunal populations (phospholipid
fatty acid (PLFA) profile and protozoan morphotypes), however, yielded a somewhat more complex picture. For the three highest Cu2þ
amendments (160, 400 and 1000 mg g21), there still was a significant reduction in number of differentiable protozoan morphotypes. The
bacterial PLFA pattern suggested a shift from Gram-negative towards Gram-positive bacteria for the high amendments, a process where
protozoan grazing most likely played a significant role. The ratio of the trans/cis isomers of the 16:1v7 fatty acid indicated that Cu2þ, even at
low and medium concentrations, induced physiological changes in the microbial population. The relatively slight changes in total microbial
and micro-faunal abundance and activity, also at the highest Cu2þ concentrations, probably reflected the ability of the community to
compensate for loss of taxa by functional substitution.
q 2003 Elsevier Ltd. All rights reserved.
Keywords: Copper; Protozoa; Bacteria; Succession; Phospholipid fatty acid; Grazing
1. Introduction
Heavy metals including copper are a potential problem in
agricultural soil because they are supplied to the soil along
with pig slurry (Christie and Beattie, 1989), sludge from
wastewater treatment plants (Tabatabai, 1976) or pesticides
(Vasseur et al., 1988). Industrial wastes may locally cause
extremely high concentrations in soil (Giller et al., 1998;
Tabatabai, 1976). There is an increasing body of evidence
suggesting that microorganisms are far more sensitive to
heavy metal stress than plants and animals inhabiting the
polluted soils (Giller et al., 1998). Heavy metal impact on
microorganisms may include decreases in microbial biomass and changes in community structure, reduced
symbiotic N2-fixation in legumes (McGrath, 1994) and a
reduced carbon mineralization, which may ultimately lead
to accumulation of deep layers of organic matter (Giller
et al., 1998).
* Corresponding author. Tel.: þ 45-3532-1275; fax: þ 45-3532-1250.
E-mail address: [email protected] (F. Ekelund).
0038-0717/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0038-0717(03)00249-9
Considering the severe effects heavy metals may have on
soil ecosystems and the important role protozoa play in soil
ecosystems as regulators of the bacterial and fungal
populations (Ekelund, 1998; Ekelund and Rønn, 1994),
the literature on heavy metal effects on soil protozoa is
surprisingly sparse. Kandeler et al. (1992) observed effects
on testate amoebae both in terms of number of species and
abundance in soil influenced by copper emitted from a
smelter, whereas Boon et al. (1998) saw no effect on
protozoan numbers in a soil heavily polluted with copper.
Most studies on heavy metals and soil protozoa have been
performed on ciliates, often involving only a few species
(Campbell et al., 1997; Forge et al., 1993; Nicolau et al.,
1999; Pratt et al., 1997); see Foissner (1999) for more details
on this subject.
Many soil microorganisms cannot be cultivated, which
impedes our knowledge on microbial diversity in soil
(Torsvik et al., 1990). Similarly, a large fraction of the soil
protozoa remains undescribed (Foissner, 1999), although,
most can be assigned to genus level. Still, neither for
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bacteria, fungi nor for protozoa, are we able to link the large
taxonomic diversity in soil to the functioning of the
microbial ecosystems (Griffiths et al., 2000). It is, e.g. not
established if a certain level of protozoan diversity is
required to ensure maximal performance, and if all groups
have to be present in order to sustain it. Neither is it known
which role protozoa play when microbial communities
recover after soil perturbation (Coûteaux and Darbyshire,
1998). A more thorough understanding of these mechanisms
would be helpful when trying to evaluate the vulnerability
of microbial ecosystems upon contamination with toxic
compounds (e.g. heavy metals).
In the present study, we used a microcosm approach to
study interactions between the protozoan grazers and their
potential food items, with major focus on the bacteria, along
a broad range of Cu2þ concentration in soil. Samples were
taken 2, 7, 14 and 70 days after the Cu2þ amendments to
ensure that the population dynamics of main microbial
groups could be monitored over a relatively large time span
to reveal their growth dynamics when the amount of
bioavailable Cu2þ supposedly had reached a stable level.
We measured protozoan abundance and diversity by
conventional microscopy. The phospholipid fatty acid
(PLFA) technique was used to estimate the in situ structure
and size of the entire microbial population. The accumulated and substrate-induced respiration (SIR) were used to
estimate microbial activity and population size,
respectively. Using this broad array of techniques we were
particularly interested in answering the following questions:
(1) At which levels in the soil will Cu2þ perturb the
microbial population? (2) How are size and diversity of the
protozoan population related to an increasing Cu 2þ
concentration and can changes in the other microbial
populations be assigned hereto? (3) To which extent will
microbial populations return towards their original structure
(resilience) after a Cu2þ perturbation event?
Table 1
Important characteristics of the clay loam soil (Schroeder, 1984; British
system) used in the experiments
Coarse sand (.200 mm)
Fine sand (20– 200 mm)
Silt (2–20 mm)
Clay (,2 mm)
Organic carbon
pH
Water holding capacity
69%
21.7%
2.8%
4.1%
1.2%
6.5
15%
conditions. Important soil characteristics are presented in
Table 1.
2.2. Microcosm setup and sampling
The soil was mixed thoroughly with finely ground barley
straw (1.125 mg straw g21 soil dw). Soil microcosms were
then prepared in 116-ml serum flasks with 10.0 g soil and
3 ml distilled water (corresponding to a soil dry matter
content of about 77%), which contained CuCl2 to give each
of 10 final Cu2þ concentrations: 0, 0.7, 1.6, 4.1, 10, 25, 64,
160, 400 and 1000 mg Cu2þ kg21 dry soil. After inoculation, the microcosms were sealed with airtight rubber
stoppers and incubated in darkness at 10 8C. On day 2, 7, 14
and 70 after start of incubation three replicate microcosms
from each Cu2þ level were destructively sampled. Accumulated CO2 in each flask was determined by injecting 1 ml
from its headspace air into a gas chromatograph with a
thermal conductivity detector (Mikrolab, Århus, Denmark).
Results from our laboratory have shown that variation in
atmospheric contents of CO2 and O2, within the limits
observed in this experiment, have no significant effects on
microbial growth (Christensen and Ekelund unpublished
results). Wet soil samples, each of 7.0 g, was taken out for
subsequent analysis of SIR and PLFA profiles (see below).
2.3. Measurements of protozoa
2. Materials and methods
2.1. Soil
A sandy soil (Schroeder, 1984; British system), sampled
from the top layer of a conventionally managed agricultural
field (Jyndevad, Denmark), was air dried, homogenised and
sieved (, 2 mm). We used a procedure with dry soil to
ensure that we observed effects on an active microbial
community. Especially for the soil protozoa it is generally
accepted that they are only active in short periods just after
soil rewetting (Clarholm, 1981, 1989; Ekelund and Rønn,
1994; Ekelund et al., 2002; Foissner, 1987) and that active
protozoa are more susceptible to the effect of xenobiotics
than non-active encysted forms (Ekelund et al., 1994). If the
treatments have only slight effects under these worst-case
conditions, their effects will probably be slight under all
We added 100 ml ‘Modified Neff’s amoeba saline’
(Page, 1988) to the remaining soil in each flask and agitated
the soil suspensions for half an hour on a ‘Heidolph Promax
2020’ end to end shaker (Struers Kebo Lab ag., Denmark) at
390 rev min21. The soil suspensions were used to prepare
microtiter plates for enumeration of bacterivorous protozoa
(Ekelund, 1999). An 0.3 g l21 autoclaved solution of
Tryptic Soy Broth (Difco Bactow, Detroit, Michigan,
USA) in Modified Neff’s amoeba saline was used as growth
medium in the microtiter plates. Data from microtiter plates
were converted to protozoan numbers by a computer
programme as detailed in Rønn et al., (1995). Protozoan
diversity (number of different discernable morphotypes) for
each treatment was determined as described by Ekelund
(1999). Heterotrophic flagellates were assigned to the
lowest taxonomic level possible, by an examination in
the microscope. The presence of ciliates, naked and testate
F. Ekelund et al. / Soil Biology & Biochemistry 35 (2003) 1507–1516
1509
amoebae, and forms with heliozoan morphology were also
recorded.
Gram-negative bacteria (18:1v7, cy17:0, cy19:0) (Wilkinson, 1988), fungi (PLFA 18:2v6,9) (Federle, 1986).
2.4. Substrate-induced respiration
2.6. Statistics
The SIR assay (Anderson and Domsch, 1978) was
performed on 300-mg portions of soil that were placed in 3ml Venoject tubes and supplied with 4 mg of a glucose and
talcum mixture (1/4, w/w) (Anderson and Domsch, 1985),
corresponding to about 3 mg glucose g21 of dry soil. After
thorough mixing the Venojects were capped, incubated in
the dark at 20 8C for 30 min and flushed for 1 min with a
technical gas (79% N2 and 21% O2) to remove any CO2.
Headspace gas was sampled 1.5 and 3 h after the initial
flushing and analysed for its concentration of respired CO2
on a Hewlett Packard 6890 GC equipped with a Porapack-Q
column and a thermal conductivity detector at an oven
temperature of 30 8C. The concentration of CO2 in the
headspace was determined by comparing the obtained areas
to a CO2 standard curve.
Data were statistically treated using two way ANOVA’s
on log10 transformed data (SigmaStatw for Windows V2.03
package). Principal component analysis (PCA) was performed on the entire PLFA data set after log10 transformation using the Unscrambler software (CAMO ASA,
Norway).
2.5. Phospholipid fatty acid analysis
Microbial biomass and community structure were
assessed using PLFA analysis. The procedure for extraction
of PLFAs was modified after Frostegård et al. (1993). Soil
samples were extracted with a mixture of chloroform,
methanol and 0.15 M citrate buffer (pH 4.0) in the
proportion 1/2/0.8 (v/v/v). The pooled supernatants from
two repeated extractions were split in two phases by
addition of chloroform and citrate buffer, after which the
lipid-containing phase was transferred to burned glass tubes
and evaporated under streaming N2. Classes of lipids were
separated on a silicic acid (100 – 200 mesh size, Sigma)
column, and the PLFAs were retained and dried under N2.
Methyl nonadecanoate was added as an internal standard
and the PLFAs were subsequently derivatised by mild
alkaline methanolysis (Dowling et al., 1986).
Samples were analysed for their content of fatty-acid
methyl esters on a Hewlett Packard 6890 GC equipped with
an auto injector, a flame ionisation detector and a 60 m HP5
(5% phenyl methyl siloxane) capillary column. Hydrogen
was used as the carrier gas (2 ml min21) and injections were
made in split-less mode. The initial oven temperature was
held at 80 8C for 5 min, increased at 20 8C min21 to 160 8C
and increased at 5 8C min21 to 270 8C, at which level it was
maintained in 5 min. Inlet and detector temperatures were
230 and 270 8C, respectively. Identification of fatty acids
was performed as in Frostegård et al. (1993) and by
comparison to the retention time of known fatty acids.
Nomenclature of fatty acid follows Tunlid and White
(1992). Attempts were made to estimate proportions of the
following main groupings of the microbial population in
samples by using PLFAs (nmol per unit dry soil) indicative
of these groups: Gram-positive bacteria (PLFAs i15:0,
a15:0, i16:0, i17:0, a17:0) (O’Leary and Wilkinson, 1988),
3. Results
3.1. Respiration
Cu2þ had a significant effect on accumulation of CO2 in
the examined soil (Fig. 1, P , 0:001; two way ANOVA on
log transformed data). During the whole experiment,
treatments with the three highest Cu2þ concentrations
differed significantly from the unamended control, whereas
the respiration in general was unaffected at the lowest Cu2þ
concentrations (P , 0:05; Tukey two way comparison).
Cu2þ as well as time had a significant effect on the
microbial biomass calculated from the SIR (Fig. 2,
P , 0:001; two way ANOVA). Likewise, we observed a
significant interaction between the two factors (P , 0:05).
At day 7, the 400 and 1000 mg g21 treatments showed a
significantly lower microbial biomass than the control
(P , 0:05; Tukey two way comparison); although not
consistently significant, this tendency was also observed at
the highest Cu2þ concentrations at the two later harvests.
3.2. PLFAs
Cu2þ concentration and time, as well as the interaction
between the factors, influenced the soil content of total
microbial PLFA significantly (P , 0:01; two way
ANOVA). The soil content of total microbial PLFA
(Fig. 3a) was similar regardless of the Cu2þ concentrations
at day 2, 7 and 70; on day 14, however, the unamended
control soil contained significantly more total microbial
PLFA than in any of the other treatments (P , 0:01; Tukey
pairwise comparison). This pattern was also observed for
PLFAs typically found in Gram-negative bacteria (Fig. 3b).
PLFAs typically found in Gram-positive bacteria
(Fig. 3c) were significantly affected by the concentration
of Cu2þ (P , 0:05; two way ANOVA), and a significant
interaction between day and concentration (P , 0:001) was
likewise observed, whereas no significant change with time
was observed. Gram-positive PLFAs were measured in
similar amounts for all Cu2þ treatments at day 2, while at
day 7 this value was increased ca. 100% in the controls and
decreased by 30 –40% in the 10 and 160 mg g21 treatments.
This pattern was even more pronounced at day 14. At day 70
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Fig. 1. Accumulated CO2 in microcosms amended with 10 individual concentrations of Cu2þ and harvested after 2, 7, 14 and 70 days of incubation. Points
represent average values (n ¼ 3). Bars show SE. Points with similar letters are not significantly different (P , 0:05; Tukey two way comparison).
the concentration of Gram-positive PLFA in the unamended
controls again dropped to the level observed at the start of
the experiment, while in the treatments with 10 –
1000 mg g21 Cu2þ it increased with increasing Cu2þ
concentrations compared to the day-2 values.
As for the bacterial PLFAs, there was a significant
variation between fungal PLFAs depending on both day
and Cu2þ concentration (P , 0:05; two way ANOVA).
Still, the fungal PLFA pattern, (Fig. 3d) differed somewhat from the pattern observed for the main bacterial
groups. The concentrations were not significantly different
at day 2, although a tendency for a decrease was seen in
the treatments with the two highest Cu2þ concentrations.
Between day 2 and 7 the fungal PLFA content increased
slightly for all treatments, though significantly only for
the 1000 mg g21 amendment (P , 0:005). The fungal
PLFAs in the unamended control continued to increase
until day 14. On day 70 all treatments contained
significantly more fungal PLFA than on day 2
(P , 0:001) with the most pronounced increase at the
highest Cu2þ concentration.
Subjecting all PLFAs to a PCA showed that at day 2
the 1000 mg g21 treatment was located apart from the rest
of the treatments that grouped together close to the origin
(Fig. 4). At day 7 and 14, the 0 and 1.6 mg g21 treatments
were located in the upper part of the diagram and visa
versa for the 160 and 1000 mg g21. After 70 days the
system returned towards the original status, as observed at
day 2, except for the 1000 mg g21 treatment, indicating
Fig. 2. Substrate-induced respiration in microcosms amended with 10
individual concentrations of Cu2þ and harvested after 7, 14 and 70 days of
incubation. Points represent average values (n ¼ 3). Bars show SE. Points
with similar letters are not significantly different (P , 0:05; Tukey two way
comparison).
F. Ekelund et al. / Soil Biology & Biochemistry 35 (2003) 1507–1516
1511
Fig. 3. Amounts of phospholipid fatty acids (PLFA) from the total microbial population (a) or typically appearing only in Gram-negative bacteria (b), Grampositive bacteria (c) and fungi (d) in microcosms. The soil was amended with 10 individual Cu2þ concentrations and microcosms were harvested after 2, 7, 14
and 70 days of incubation. Each data point represents the average (n ¼ 3; bars indicate SE).
that the microbial community structure was permanently
altered only at the highest Cu2þ concentration. The
overall pattern throughout the experiment showed a
continuous distribution (from 0 to 1000 mg g21) of the
values along the ordinate (from top to bottom), indicating
that PC2 represented the variation arising directly from
the Cu2þ treatments. Although PC1 contributed to a large
part of the variation (78%), a direct connection to the
Cu2þ concentrations was not observed. In the loading plot
(data not shown) typical PLFAs from Gram-positive
(i15:0, a15:0) and Gram-negative (18:1v7) bacteria
contributed considerably to the variation in each direction
along the PC1-axis.
3.3. Trans/cis ratio of PLFA 16:1v7
The trans/cis ratio of PLFA 16:1v7 (Fig. 5) was affected
by time and Cu2þ concentrations (both P , 0:0001; two
way ANOVA), which also resulted in a significant
interaction (P , 0:001). Through the 70 days the trans/cis
ratio in the 0, 160 and 1000 mg g21 treatments was stable,
although decreased by about 35% at the two highest
concentrations. Contrasting, in the 1.6 and 10 mg g21
treatments the trans/cis ratio decreased dynamically through
the first 14 days followed by an increase towards the end of
the experiment, this development being strongest at
10 mg g21.
Fig. 4. Score plots of the two first components (PC) in a principal
component analysis of the mol% of microbial PLFAs in the microcosms.
The soil was amended with 10 individual Cu2þ concentrations and
microcosms were harvested after 2, 7, 14 and 70 days of incubation. The
calculation included all measured microbial PLFAs in all four harvests,
however, the data from the individual harvests are presented in each of four
panels for reasons of clarity. Each data point represents the average (n ¼ 3;
bars indicate SE) of the coordinate values within each treatment at the
individual sampling events.
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The number of discernible morphotypes was significantly affected by addition of Cu2þ on all sampling dates
(Fig. 7, P , 0:001; one way ANOVA). Throughout the
experiment, the protozoan diversity for the three highest
Cu2þ concentrations remained lower than in the other
treatments and significantly (P , 0:05; Tukey two way
comparison) different from the unamended control. Only
Heteromita globosa, Cercomonas minimus, and naked
amoebae were able to withstand 1000 mg g21 Cu2þ, and
there was a clear tendency that some forms only occurred in
samples with a low Cu2þ addition (data not shown).
4. Discussion
Fig. 5. Trans/cis ratio of the16:1v7 phospholipid fatty acid (PLFA)
extracted from soil microcosms amended with 0, 1.6, 10, 160 and
1000 mg g21 Cu2þ and harvested after 2, 7, 14 and 70 days of incubation.
Each data point represents the average (n ¼ 3; bars indicate SE). Points
with similar letters are not significantly different (P , 0:05; Tukey two way
comparison).
3.4. Protozoa
Total protozoan abundance (Fig. 6) was affected
significantly (P , 0:01; two way ANOVA on log transformed data), but to a much smaller extent than the other
properties. No significant differences in protozoan abundance between concentrations were seen after day 7.
4.1. Microbial activity
We amended the soil with barley straw in order to mimic
a post-harvest situation, where plant residues induce a high
microbial activity (Christensen et al., 1992; Rønn et al.,
1996). The large amount of CO2 released in the microcosms
during the experimental period (Fig. 1) demonstrates that
the straw material did stimulate the microbial activity. In the
treatments with no or low Cu2þ amendment the total amount
of carbon mineralised during the 70 days made up about
60% of the original amount of C applied as straw, whereas
only about 30% was mineralised in the 1000 mg g21
Fig. 6. Protozoan abundance (MPN) g21 dry soil (log) recovered from microcosms amended with 10 individual concentrations of Cu2þ and harvested after 2, 7,
14 and 70 days of incubation. Points represent average values (n ¼ 3). Bars show SE. Points with similar letters are not significantly different (P , 0:05; Tukey
two way comparison).
F. Ekelund et al. / Soil Biology & Biochemistry 35 (2003) 1507–1516
1513
Fig. 7. Protozoan diversity measured as number of ‘morphotypes’ recorded from microcosms amended with straw and various concentrations of Cu2þ. Highly
significant differences (P , 0:001; one way ANOVA, two way ANOVA abandoned due to violation of test conditions) of Cu2þ addition were found on every
sampling occasion. Columns represent average values (n ¼ 3). Bars show SE. Bars without similar letters represent significantly different values (P , 0:05;
Tukey two way comparison).
treatment. Thus, at high concentrations, Cu2þ had a strong
negative effect on the microbial mineralization performance. Likewise, the microbial biomass, determined as SIR,
was reduced at the highest Cu2þ concentrations (Fig. 2).
This estimate of biomass did not correlate with the content
of total PLFAs in the soil. However, where the SIR
probably measures the active, and to a lesser degree the
inactive part of the microbial population (Anderson and
Domsch, 1978), the PLFA method includes the entire
microbial population (Tunlid and White, 1992). Choosing a
total PLFA concentration of 30 nmol g21 dry soil and a
carbon content of 17 C (calculated weighed mean) atoms
per fatty acid, this corresponds to approximately
6 mg C g21 dry soil in microbial membrane fatty acids –
or 2% of the biomass C if using a SIR value of 300 mg g21
dry soil. Similar amounts of PLFA C relative to total
cellular C have been observed in bacteria (Balkwill et al.,
1988) and fungi (Klamer and Bååth, pers. comm.).
Although these calculations should be taken with precaution, they indicate that the present SIR and PLFA estimates
are within comparable ranges.
4.2. Protozoa
Both abundance and diversity of the protozoa in the soil
microcosms were affected by the Cu2þ amendments
(Figs. 6 and 7). Total protozoan abundance, though, was
only affected significantly at day 2 and 7. The pattern on
day 2 probably reflects a dose-related protozoan death,
whereas the day-7 pattern reflected that growth was
inhibited also for relatively small concentrations, supporting previous findings that protozoa are most susceptible to
toxic compounds during growth (Ekelund et al., 1994).
Further ahead in the succession, the more Cu2þ tolerant
strains were probably favoured, blurring the toxic effect on
the system. We attribute the apparent tendency to an
inverse dose-response relation on day 70 to growth of
small opportunistic forms, a suggestion, which is also
supported by the actual observed composition of morphotypes (data not shown). The effect on the protozoan
diversity was much more pronounced and the number of
morphotypes was lowered at the three highest Cu2þ
concentrations. A stronger effect on diversity than on
abundance may be expected, since some protozoa are more
susceptible to certain toxic xenobiotics than others
(Ekelund, 1999; Ekelund et al., 2000; Griffiths et al.,
2000). Implementation of other techniques for the study of
protozoan diversity would probably have allowed discovery of additional protozoan types like the autochthonous
forms (Petz et al., 1985, 1986) and might have revealed
even larger differences between Cu2þ-amended and
unamended systems.
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4.3. PLFA patterns
The changes in population biomass levels as indicated by
the PLFA plots (Fig. 3a – d) probably reflect dynamic
steady-state situations governed by input from microbial
growth and removal by micro-faunal grazing in combination with toxic effects of the applied Cu2þ. In the
unamended systems, total microbial, Gram-negative and
Gram-positive PLFA (Fig. 3a – c) increased to a certain
level, after which the grazing exceeded the growth and the
populations declined again. Increasing the Cu2þ level to 1.6
and 10 mg g21 led to inhibition of the microbial growth,
while the protozoa were left unaffected, which resulted in a
reduced population level at day 14, where the unamended
systems peaked. For the 160 and 1000 mg g21 treatments,
effects on the protozoan community structure (Fig. 7)
caused a less efficient grazing performance, thus allowing
growth of microorganisms with a high Cu2þ tolerance
experiencing a diminished grazing pressure. This is
supported by the fact, that many protozoa have relative
specific food preferences (Ekelund and Rønn, 1994; Rønn
et al., 2001). Comparison of the PLFA patterns of the Gramnegative and Gram-positive bacteria (Fig. 3b and c),
strongly suggests that the protozoan types surviving the
high Cu2þ concentrations for more than 14 days preferred to
prey on Gram-negative bacteria. Their PLFA value was
nearly halved from day 14 and on, while the opposite pattern
was seen for Gram-positive bacteria.
The fungal PLFA graphs (Fig. 3d) indicate that grazing
pressure on fungi was insignificant in this study, which is
probably related to the generally rather small populations of
fungivorous protozoa in soil (Ekelund, 1998; Hekman et al.,
1992). Some nematodes likewise graze on fungi, but they
probably played an insignificant role here, as their ability to
colonise a substrate depends on migration from a large
surrounding soil volume (Griffiths and Caul, 1993) not
present in this study. Chander et al. (2001) and Huysman
et al. (1994) found indications that heavy metal contamination enhanced the proportion of fungal biomass relative to
the bacterial, as in our experiment. The enhanced fungal
population late in the experiment were probably also able to
exploit the recalcitrant straw residues better than the
bacteria.
The PCA plots show the most important trends in the
variation in the entire fatty acid data set and it is clear that
the two highest Cu2þ concentrations affected the microbial
community differently than the 0 and 1.6 mg g21 treatments.
The effect of the 1000 mg g21 treatment was evident already
after 2 days, indicating that this load of Cu2þ had a direct
toxic effect on the microorganisms. Moreover, the community structure seemed permanently changed only at
1000 mg g21 as its data point never grouped up with the
other treatments, which seemed to return to the original
population structure.
Pure-culture studies have shown that microorganisms
change the proportion of membrane trans fatty acids relative
to the respective cis isomers as a way to cope with various
changes in their life conditions (Guckert et al., 1986;
Heipieper et al., 1994, 1996). The microorganisms use the
trans/cis ratio to adjust the fluidity of the membrane. We
observed that Cu2þ amendment led to a significant decrease
in the trans/cis ratio (Fig. 5), for the 1.6 and 10 mg g21 Cu2þ
treatments. It appears that at low concentrations, Cu2þ
interacts with the cell membrane phospholipids and thereby
decreases its fluidity (Suwalsky et al., 1998). Under such
conditions, the living cells may decrease their trans fatty
acid content in order to keep the fluidity at a proper level.
4.4. Conclusion
Cu2þ added to soil affected both bacteria and protozoa
regarding activity, physiology and diversity. Except for the
highest concentrations, this effect was transient although
Gram-positive bacteria seemed to be more directly affected
by the Cu2þ than Gram-negative bacteria. The relative slight
changes in total microbial and micro-faunal abundance and
activity, even at high levels of Cu2þ, probably reflect an
ability of the microbial community to quickly compensate
for loss of taxa by functional substitution. The return of the
community to a PLFA composition close to the origin after
70 days (except for the highest Cu2þ concentrations) could
be indicative of such a substitution. The apparent permanent
changes in the number of protozoan taxa groups and
microbial PLFA profiles, suggest that absorption of Cu2þ to
soil particles did not happen to an extent that could alleviate
the toxic effects of Cu2þ even within this relatively long
period of time.
Acknowledgements
F.E. and A.J. were supported by the Danish Environmental Research Programme (Centre for Biological
Processes in Contaminated Soil and Sediment and Centre
for Effects and Risks of Biotechnology in Agriculture,
respectively). F.E. acknowledges support from the Danish
Natural Science Research Council. The competent assistance of lab technician Ulla Rasmussen is greatly
appreciated.
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