MICROBIAL

ECOLOGY
Microb Ecol (2002) 44:49-58
DOh 10.1007/s00248-001-0042-8
2002 Springer-Verlag New York Inc.

The Diversity and Function of Soil Microbial Communities

Exposed to Different Disturbances
A.K, Miiller,1 K. Westergaard, 1 S. Christensen, 2 S.J. Sorensen 1
1 Department of General Microbiology, University of Copenhagen, Solvgade 83 H, DK-1307 Copenhagen K,
Denmark
2 Department of Terrestrial Ecology, University of Copenhagen, Universitetsparken 5, DK-2100
Copenhagen O, Denmark
Received: 2 May 2001; Accepted: 15 October 2001; Online publication: 30 April 2002

IA

B ST R A C T

To improve understanding of the relationship between the diversity and function of the soil
ecosystem, we investigated the effect of two different disturbances on soil bacterial communit i e s - l o n g - t e r m exposure to the heavy metal mercury and transient exposure to the antibiotic
tylosin. In the mercury-contaminated soil the diversity (Shannon index) was reduced as assessed
from denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the
soil community DNA and from colony morphology typing of the culturable bacterial population.
However, analysis of the substrate utilization profiles did not reveal any differences in diversity.
In the tylosin-treated soil, DGGE revealed a small difference in the diversity of 16S rDNA
compared to the control soil, whereas analysis of the colony morphology typing or substrate
utilization results did not reveal any differences in diversity. Soil function was also affected b y
mercury contamination. The lag time before soil respiration increased following addition of
glucose or alfalfa substrate was longer in the mercury-contaminated soil than in the control soil.
Moreover, it was markedly prolonged in mercury-contaminated soil subjected to heat treatment
prior to substrate addition, thus indicating reduced resistance to a new disturbance in the
mercury-contaminated soil as compared to the control soil. Tylosin treatment did not have any
significant effect on any of the respiration parameters measured, either with or without prior
heat treatment of the soil.

Introduction

of theories have been formulated concerning how species

d i v e r s i t y is r e l a t e d to e c o s y s t e m f u n c t i o n [31]. T h u s , N a -

T h e r e is g r o w i n g i n t e r e s t in the r e l a t i o n s h i p s a m o n g ec-

e e m et al. [39] a n d T i l m a n et al. [53] have s u g g e s t e d t h a t

o s y s t e m d i v e r s i t y , s t r u c t u r e , a n d function, a n d a n u m b e r

enhanced

species d i v e r s i t y is beneficial to

function.

In contrast,

Correspondence to: S.J. Sorensen; Phone: +45 35 32 20 53; Fax: +45 35 32

20 40; E-maii: sjs@mermaid.molbio.ku.dk

other

authors

ecosystem

suggest that

the

properties of an ecosystem depend more upon the func-

50
tional abilities of particular species than on the total
number of species [28, 54, 58].
One aspect of ecosystem function is stability, defined as
the system's ability to avoid displacement following a
perturbation (resistance) and to return to its former state
following a perturbation (resilience) [5]. Stability has
formerly and more recently been suggested to correlate
positively with system diversity [12, 20, 33, 52], although
the validity of this hypothesis is the subject of controversy
[22].
Despite the suggestion of Wardle and Giller [57] that
the diversity and functional importance of soil organisms
present an excellent opportunity to test currently topical
aspects of ecological theory, the hypotheses relating ecosystem diversity and function have mainly been developed
by plant ecologists, with only a few studies having concerned the soil community [21, 35]. Hypotheses from the
aboveground communities may not easily be applied to
belowground, since there are differences. The microbial
diversity in soil is enormous [55], and there may be substantial overlap in function between microbial species [9].
Furthermore, it is likely that microorganisms within a
functional group differ in their response to the environment. As microorganisms are fast growing they can
quickly fill out empty niches occurring when the environment is changing [19]. These circumstances could
create a high degree of stability, but it is unknown what
level of diversity is necessary to maintain stability [57].
Investigation of the relationships among diversity,
structure, and function poses the problem of how to
manipulate communities so as to establish differences in
diversity for comparative purposes. One possibility is to
construct communities differing in level of diversity by
introducing different numbers of species into microcosms [34, 35]. However, such experiments almost inevitably involve unrealistically few species within the
communities. Moreover, on the microbial level a created
community will be unrepresentative of a natural community since only known and culturable organisms can
be included. They are unlikely to cover important functions in the soil system. Another approach is destructive
reduction of species diversity [21, 47]. This inevitably
entails selective reduction in diversity, and if species
vulnerable to the treatment are removed the results may
more reflect the absence of certain key species than a
reduction in diversity. However, this is also the case in
natural systems, where any given stress always selects for
adapted species. The results of studies made using the

A.K. M/iller et al.

destructive approach will gradually become more interesting when different means of reducing diversity with
different selective properties are compared.
The effect of a disturbance on microbial community
function depends on its duration and specificity. After a
transient disturbance, system function may eventually
return to its former state, whereas a permanent disturbance will result in a new, altered state [46]. Disturbances
with a specific mode of action only alter a few groups of
organisms, whereas those that act non-specifically affect a
wide range of organisms. Investigation of how different
kinds of disturbances affect system function will enhance
our knowledge of the relationships among diversity,
structure, and function.
Heavy metals are highly persistent in the environment
and are known to alter soil ecosystem diversity, structure,
and function [7, 18]. Antibiotics dispersed into the soil in
connection with the application of livestock manure represent transient disturbances that mainly affect specific
groups of bacteria. Although the effects of antibiotics in
soil have been investigated [6, 16, 29], their effect on the
structure and function of the microbial community has
not previously been examined.
The diversity of complex soil microbial communities
has been studied using new techniques for describing
different microbial populations [8, 17, 37, 51, 56]. As no
one single method is currently available for exploring the
whole bacterial community, a combination of methods is
necessary to obtain a detailed view of its structure and
diversity.
When characterizing system function a myriad of different processes can be examined. The key soil function is
mineral cycling, and one of the most common measures of
microbial mineralization is respiration [44].
The aim of the present paper is to investigate how two
different disturbances--mercury contamination and tylosin treatment--affect the diversity and function of the soil
microbial community. Community diversity was assessed
by colony morphology typing, DGGE profiling, and substrate utilization studies. For both soils detailed results
from these community analyses have already been published [38, 60], and here we only present diversity as the
Shannon Index. Community function was assessed from
CO2 production following substrate addition (glucose or
alfalfa). In order to investigate whether the resistance of
the soil microbial community was compromised by the
initial disturbance, the mercury-contaminated and tylosintreated soils were also subjected to an additional distur-

Disturbed Microbial Communities

b a n c e (heat t r e a t m e n t )

p r i o r to the m e a s u r e m e n t of

respiration.

Materials and Methods

Tylosin Treatment
Soil samples were collected from the upper 20 cm of an agricultural sandy soil in Jyndevad, Southern Jutland, Denmark. The
mineral fraction of the soil from this location consisted of 4.1%
clay (<2 lain), 2.8% silt (2-20 ~tm), 21.7% fine sand (20-200 ~tm),
and 69% coarse sand (>200 ~m). The soil contained 1.2% organic
carbon and had a water-holding capacity of 15 g water 100 g-1 of
soil. The pH was 6.8 [27]. After sampling the soil was brought to
the laboratory and stored at 10C in the dark until required. The
soil was mixed, sieved (2 ram), air-dried overnight at room
temperature, and transferred to 8 plastic boxes (750 g soil in
each). Tylosin-treated soils (four replicate soil boxes) were prepared by dissolving tylosin (tylosin tartrate, Sigma) in water and
thoroughly mixing the solution into the soil to a final concentration of 2000 ~tg tylosin g-Z dry soil and a water content of 15%
(corresponding to the water-holding capacity of the soil). The
remaining four soil boxes served as controls, with only water
being added. The soil was incubated aerobically at 25C in the
dark. On day 60, when the number of CFU in the tylosin-treated
soils was no longer significantly different from that in the controls (t-test, p < 0.05), the soil boxes were transferred to 4C. It is
unknown whether the proportion of culturable bacteria in the
treated and control soil may have differed. The community
analyses were made on each of the four tylosin-treated and
control boxes of soil.

51
trifuged for 2 min at 10,000 g, and the supernatant was collected in a new tube. The procedure was then repeated twice
on the pellet. Finally, the 3 mL supernatant was mixed
thoroughly and filtered through a 0.2 ~tm cellulose acetate
filter.
Samples were analyzed by HPLC (LKB Bromma, 2248, Pharmacia, Uppsala, Sweden) equipped with an RP C-18 column (5
~tm particle size) and a variable-wavelength monitor (LKB
Bromma, 2248, Pharmacia, Uppsala, Sweden) using the mobile
phase as described by Ose and Tonkinson [42]. Peaks were detected at 280 nm. The standard curve was made from analyzing
standard solutions of different concentrations of tylosin tartrate
in distilled H20.

Quantification of Total and Bioavailable Mercury

The total mercury content of the soil was determined using
static headspace analysis as previously described by Kriger and
Turner [30] (soil method 2) whereby the Hg present is converted to the elemental form and measured on a Hg vapor analyzer (Jerome Model 431-X, Arizona Instruments Inc., Phoenix,
AZ).
The bioavailable inorganic mercury was measured using a
mer-lux biosensor as previously described [45] combined with
calculation of the mer-lux expression factors [4]. A standard
curve was established using the regression equation for the relationship between the amount of bioavailable mercury and the
expression factors obtained from a standard assay (Hg(NO3)2
diluted in distilled H20). A control experiment was performed
using a mutant strain constitutively expressing the lux genes [3]
to investigate whether inhibition of light development occurred
in soil samples.

Mercury-Contaminated Soil
Soil samples were collected from the upper 5-15 cm at a mercurycontaminated site in Assens, Denmark. The contamination with
elemental mercury had taken place 14 years prior to sampling.
The samples were collected in a distance of 1 m and 19 m from
the center of contamination in order to obtain one sample subjected to a high concentration of mercury and one sample free of
mercury contamination to serve as a control. The mercurycontaminated soil (pH 7.2) contained 4.3-6.3% organic matter
[38] and was sampled at a site devoid of vegetation. The control
soil (pH 7.0) contained 6.6-6.9% organic matter [38] and was
sampled in a garden. The samples were stored in glass jars at 4C
in the dark up to 6 months. The storage at 4C has been shown to
decrease the number of viable organisms [48]. The community
analyses were made on three replicate soil samples from each
collection point.

Quantification of Tylosin by HPLC

One g soil was mixed with 1 mL distilled H20 in an Eppendorf
tube. The solution was shaken vigorously for 1 min and cen-

Bacterial Colony Morphology

Soil was diluted 10-fold in phosphate-buffered saline (PBS;
autoclaved solution of 1.44 g L-1 Na2HPO4-2H20, 0.24 g L-~
KH2PO4, 8.00 g L-I NaC1, and 0.20 g L-I KC1 in distilled water)
and vortexed at maximum velocity for 1 rain. From this suspension, soil dilutions appropriate to ensure 30 to 100 colonies
per plate were spread on TSA (Tryptic Soy Agar) plates supplemented with fungicide (25 ~tg natamycin mL-1). The plates were
inspected after 4 days of incubation at 25C. A nutrient-rich
medium was used to enhance expression of the differences between colonies [43]. The colonies were grouped into morphotypes on the basis of visual differences according to Smibert and
Krieg [50] using characteristics such as colony color, diameter,
edge, surface (roughness and shininess), and other special
characteristics, e.g., tendency to swarm. Two hundred random
colonies were typed on each replicate of the tylosin-treated and
control soils while 150 colonies were typed on the mercurycontaminated soil. The number of different colony morphotypes
and the number of colonies belonging to each morphotype were
used as parameters for further analysis.

52

DNA Fingerprinting
Microbial community DNA from the soils used in the tylosin
experiment was extracted using a bead-beating method described
by van Elsas and Smalla [ 11]. Extracts were initially purified with
CsC1 and thereafter using the Wizard DNA cleanup system
(Promega, Madison, WI) in accordance with the manufacturer's
instructions and then stored at -20C. Microbial community
DNA from the mercury-contaminated soil was extracted using
the bead-beating method (FastDNA SPIN Kit (for soil), Bio 101
Inc., USA) in accordance with the manufacturer's instructions.
The extract was further purified by Wizard DNA cleanup system
and then stored at -20C.
The community DNA extracted from the soil was amplified
using the primer sequences described by Muyzer et al. [37].
These anneal to conserved regions of the 16S rDNA of eubacteria
and contain a GC clamp. Amplification was undertaken using the
Expand High Fidelity DNA polymerase (Boehringer Mannheim)
in accordance with the manufacturer's instructions using 250 nM
of each primer. The polymerase was added after a hotstart procedure (94C for 5 min). The PCR was then performed with a
Perkin-Elmer 9600 thermocycler using the following cycles: 1
min at 94C, 1 min at 65C, 3 rain at 72C, with a touchdown of
0.5C per cycle for the first 20 cycles. Thereafter followed 10
cycles at the annealing temperature of 55C. The tenth cycle was
followed by 7 min at 72C.
After gel electrophoresis (2% w/v agarose gel) of 5 ~L subsamples of the PCR product the amount of amplified DNA was
calculated by comparing band intensities to a standard curve
based on intensities from a Low DNA Mass Ladder (Gibco-BRL).
Comparisons were performed by image analysis using Quantity
One 4.0.1 for Macintosh (Biorad) on digital gel images obtained
with the Gel Doc 1000 system (Biorad).
DGGE of the amplified 16S rDNA sequences was performed as
described by Muyzer et al. [37] with minor modifications. Equal
amounts of DNA were loaded on the gel. Digital image of the gel
was obtained and analyzed using image analysis (Quantity One
4.0.1, Bio-Rad). By carefully inspecting lane intensity curves in
combination with enlarged images of the lanes bands were detected and quantified (average peak intensity). Background intensity was subtracted (option: Rolling disc, size 9). The number
of bands and the intensity of each band were used as parameters
for further analysis.

Sole Carbon Source Utilization Profile

Bacterial cells for inoculation of Ecoplates (Biolog Inc., Hayward,
CA) were extracted from soil by homogenizing 10 g of soil in 100
mL sterile water for 1 min in a Waring blender followed by I min
cooling on ice and further blending for 1 min. The slurry was
then centrifuged for 10 min at 1000 g, and the supernatant
transferred to a new tube. The pellet was resuspended in 100 mL
sterile water, and the blending procedure repeated. The two
supernatants were pooled and diluted to obtain a cell density
(estimated by direct count after staining with acridine orange) of

A.K. Mfiller et al.

approximately 4 x 105 cells mL -1. Extract (125 pl) was inoculated
into each well and the plates incubated in the dark on a shaker
(150 rpm) at 20C. The OD59s was measured with a microtiter
plate reader (EL 340 Biokinetics Reader, Biotek Instruments,
Winooski, Vermont, USA). In order to minimize the effects of
different inoculation densities the plates were read when the
average well color density (AWCD) on a plate was 0.5 _+ 0.1. The
data were standardized by subtracting the reading of the blank
well from the reading of each substrate-containing well and dividing this value by the AWCD [18]. Wells with a mean OD < 0
were excluded from further analysis as they do not contribute to
the biological information. The number of substrates utilized
(well color > 0.1) and the standardized color development of
each substrate were used as parameters for further analysis.

Diversity Index and PCA

The results of the three different community analyses were
evaluated by determining the Shannon diversity index. The index
based on colony morphology, DGGE, or substrate utilization was
calculated as
$

Ht = -- ~

Pi

In

Pi

i=1

where Pi is the percentage of (a) the total (S) intensity accounted

for by the ith band, (b) the total coloration accounted for by the
ith substrate, or (c) the total number of colonies accounted for by
the ith morphotype, respectively.
Statistical analysis of the results was performed by analysis of
variance. The principal component analysis was based on the
correlation matrix using SPSS 6.1 for Macintosh.

Soil Respiration Parameters

We also examined the effect of tylosin-treatment and mercurycontamination on soil respiration following the addition of
substrate. Moreover, to investigate the ability of the treated and
contaminated soil to resist a further disturbance, we measured
respiration on soil samples that had been heated in watertight
plastic tubes at 50C for 12 h prior to the addition of the substrates.
The two substrates used were (a) glucose plus ammonium
nitrate (40 mg glucose g-1 wet soil and 11.4 mg NH4NO3 g-~ wet
soil) and (b) ground alfalfa (5 nag alfalfa g-1 wet soil). The
substrate, 2.5 g soil and 10 mL sterile water were added to
flasks, which were then sealed and incubated at 15C on a
shaker.
The headspace CO2 from the glucose-amended soil samples
was measured every second hour over a 48-hour period, while
that from the alfalfa-amended soil samples was measured every 4
to 8 hours over a 72-hour period. The gas samples were analyzed
on a gas chromatograph (Mikrolaboratoriet, Aarhus, DK) with a
TC detector after separation on a 1.8 m 3 m m Porapak Q
column.

Disturbed Microbial Communities

53

The various respiration parameters determined were analyzed

statistically by analysis of variance (SAS 6.12 for Windows).

Results
Quantification of Tylosin
The added tylosin was recovered completely on day 1-3 of
incubation (data not shown). On day 5, the first of 4 unidentified peaks appeared on the chromatogram, probably
representing tylosin degradation products. After day 13,
tylosin could not be detected, and by day 17, all degradation products had disappeared. None of the abovementioned c o m p o u n d s were detected in the control soils.

Quantification of Mercury
The total m e r c u r y concentration in the soil collected 1 m
from the centre of contamination was 511 ~tg Hg g-1 dry
soil, of which only 0.043% was bioavailable.
The total m e r c u r y concentration in the control soil was
6.8 ~tg Hg g-1 dry soil, with no bioavailable mercury being
present (detection limit: 35 ~g Hg g-1 soil). Even though
the total m e r c u r y concentration was higher than expected,
it was nevertheless considerably lower than in the soil
close to the center of contamination.

Diversity of the Bacterial Community

The bacterial diversity in the various soils based on
colony m o r p h o t y p e s , DGGE, and substrate utilization is
presented in Table 1. More results from the c o m m u n i t y
analysis other than the diversity index has previously
been published [38, 60]. Among a variety of different
diversity indices [32], the Shannon index is one of the
most widely used. However, this index requires clearly
Table 1. Bacterial diversity (mean + SE) in the different soils
estimated as the Shannon index based on colony morphotypes,
DGGE bands and substrate utilization (Ecoplates)
The Shannon diversity index based on:
Treatment

Colony
morphotypes

Control
Tylosin
Control
Mercury

2.48
2.54
2.66
2.10

+
+
+
+

0.04
0.05
0.10
0.15"

DGGE bands
3.75
3.65
3.83
3.48

+
+
+
+

0.01
0.03*
0.02
0.02***

Substrate
utilization
2.62
2.54
2.66
2.64

+ 0.05
_+ 0.07
_+ 0.07
+ 0.08

Statistically significant (ANOVA) effectsof tylosin treatment and mercury

contamination are indicated as *p < 0.05 and ***p < 0.001.

defined species and a distinct identification of individuals

[59], requirements that are not met when dealing with
bacteria. Estimation of the Shannon index as p e r f o r m e d
in this investigation thus provides composite values for
the n u m b e r and distribution of morphotypes, DGGE
bands, and substrates utilized that represent different
aspects of bacterial diversity, but not necessarily at a
species level.
The Shannon index values given in Table 1 are the
average values for the replicates of each soil. Although it
has been questioned whether it is statistically correct to
apply parametric statistics to the Shannon index [14, 32],
the issue is considered to be of m i n o r importance in the
present study since the differences between the soils were
the same irrespective of whether the index was calculated
for all the separate replicates (data not shown), for the
pooled replicates (data not shown), or as the average of the
replicates (Table 1).
The Shannon indices calculated for the three m e t h o d s
of describing the microbial c o m m u n i t y are not directly
comparable since one of the parameters determining the
index is the n u m b e r of variables (in this case either the
n u m b e r of bands, substrates or morphotypes), which of
course differs between the three methods.
There was no statistically significant difference in diversity between the tylosin-treated and control soils after 2
months of incubation as estimated on the basis of colony
m o r p h o t y p e s (iV = 0.36) and substrate utilization (p =
0.37), although there was a difference in diversity as estimated on the basis of the DGGE bands (p = 0.02), mainly
due to the lower n u m b e r of DGGE bands in the treated soil
(data not shown).
With the m e r c u r y - c o n t a m i n a t e d soil, diversity differed
significantly between the contaminated and control soils
as estimated on the basis of both colony m o r p h o t y p e s (p =
0.04) and DGGE bands (p = 0.0003). In both cases, bacterial diversity was lower in the m e r c u r y - c o n t a m i n a t e d
soil than in the control soil, mainly because of differences
in the n u m b e r of m o r p h o t y p e s and the n u m b e r of DGGE
bands (data not shown). Diversity as estimated on the
basis of substrate utilization did not differ between the two
soils (p = 0.85).

Soil Respiration Parameters

The respiration parameters of soil amended with glucose
plus a m m o n i u m nitrate was the substrate-induced respiration (SIR) rate, the lag time before the exponential in-

54

A.K. M/flier et al.

c r e a s e in r e s p i r a t i o n r a t e a n d t h e specific r e s p i r a t i o n in-

c o n s i d e r a b l y i n c r e a s e d t h e d e l a y (p = 0.0001), b u t to the

c r e m e n t d u r i n g t h e e x p o n e n t i a l p h a s e [40]. As no expo-

s a m e extent in b o t h t y l o s i n - t r e a t e d a n d c o n t r o l soils. The

n e n t i a l i n c r e a s e in r e s p i r a t i o n o c c u r r e d after the a d d i t i o n

r e s p o n s e o f the two soils to h e a t t r e a t m e n t d i d differ in

o f alfalfa, o n l y two r e s p i r a t i o n p a r a m e t e r s were deter-

r e g a r d to r e s p i r a t i o n rate, h o w e v e r (p = 0.04). Thus res-

m i n e d : the t i m e i n t e r v a l b e f o r e CO2 p r o d u c t i o n s t a r t e d to

p i r a t i o n rate i n c r e a s e d following heat t r e a t m e n t in the

i n c r e a s e ( r e s p i r a t i o n d e l a y ) a n d the r e s p i r a t i o n rate. The

c o n t r o l soil b u t r e m a i n e d u n c h a n g e d in the t y l o s i n - t r e a t e d

l a t t e r was h i g h e r t h a n t h e b a s a l r e s p i r a t i o n rate.

soil.

the results o f the r e s p i r a t i o n

SIR rate in the m e r c u r y - c o n t a m i n a t e d soil d i d n o t

measurements on tylosin-treated and mercury-contami-

differ f r o m that in the c o n t r o l soil (p = 0.76), whereas

Table 2 s u m m a r i z e s

n a t e d soil a m e n d e d w i t h g l u c o s e plus a m m o n i u m nitrate

the lag time b e f o r e the e x p o n e n t i a l increase in r e s p i r a -

o r a m e n d e d with alfalfa. T h e SIR rate t e n d e d to b e h i g h e r

tion rate was s i g n i f i c a n t l y l o n g e r (p = 0.004) a n d the

(p = 0.09) a n d specific r e s p i r a t i o n i n c r e m e n t l o w e r (p =

specific r e s p i r a t i o n i n c r e m e n t l o w e r (p = 0.05). The re-

0.09) in the t y l o s i n - t r e a t e d soil t h a n in the c o n t r o l soil,

s p o n s e o f the two soils to h e a t e x p o s u r e differed. T h u s

t h u s i n d i c a t i n g a h i g h e r m i c r o b i a l b i o m a s s with a slower

the lag t i m e i n c r e a s e d in t h e c o n t a m i n a t e d soil b u t de-

g r o w t h in the t y l o s i n - t r e a t e d soil. The h i g h e r SIR rate in

c r e a s e d in the c o n t r o l soil (p = 0.05). W i t h b o t h soils,

the t y l o s i n - t r e a t e d soil c o r r e s p o n d s to e s t i m a t e s o f the

the specific r e s p i r a t i o n i n c r e m e n t was significantly re-

microbial biomass made using the fumigation-extraction

d u c e d b y the h e a t t r e a t m e n t (p = 0.01). The r e s p i r a t i o n

m e t h o d ( d a t a n o t s h o w n ) . T h e lag t i m e b e f o r e the expo-

d e l a y following the a d d i t i o n o f alfalfa was significantly

n e n t i a l i n c r e a s e in r e s p i r a t i o n rate was n o t significantly

l o n g e r in the m e r c u r y - c o n t a m i n a t e d

soil t h a n in the

affected b y the p r e s e n c e o f t y l o s i n (p = 0.45). H e a t treat-

c o n t r o l soil (p = 0.0001), a difference that was further

m e n t p r i o r to the r e s p i r a t i o n m e a s u r e m e n t s t e n d e d to

e n h a n c e d b y h e a t t r e a t m e n t . T h u s whereas there was no

i n c r e a s e the lag t i m e (p = 0.08) a n d the specific r e s p i r a t i o n

r e s p i r a t i o n d e l a y in e i t h e r t h e u n h e a t e d o r h e a t e d c o n -

i n c r e m e n t (p = 0.09), b u t w i t h n o differences b e t w e e n the

trol soil, the d e l a y in the m e r c u r y - c o n t a m i n a t e d

t y l o s i n - t r e a t e d soil a n d c o n t r o l soil. T h e SIR rate was not

d o u b l e d f r o m an average o f 22.7 h w i t h o u t heat treat-

affected b y the h e a t t r e a t m e n t (p = 0.23). Tylosin treat-

m e n t to 44 h following h e a t t r e a t m e n t . W h e n d e c o m -

m e n t h a d no effect o n t h e r e s p i r a t i o n d e l a y a n d r e s p i r a -

p o s i t i o n started in the m e r c u r y - c o n t a m i n a t e d soil, the

t i o n rate in soil a m e n d e d w i t h alfalfa. Heat t r e a t m e n t

r e s p i r a t i o n rate was

significantly higher than

soil

in the

Table 2.

Soil respiration parameters (mean + SE) calculated after addition of glucose (SIR rate, lag time and specific respiration
increment [Spec. resp. inc.]) or alfalfa (respiration delay and respiration rate) for tylosin-treated soil and mercury-contaminated soil
with (+) and without ( - ) heat treatments
Addition of alfalfa

Addition of glucose and ammonium nitrate

Soil
Control
Control
Tylosin
Tylosin
Tylosin treatment
Heat treatment
Interaction
Control
Control
Mercury
Mercury
Mercury contamination
Heat treatment
Interaction

Heat
+
+

SIR
(~tg CO2 g-t
dw soil h -1)
3.36
2.07
5.74
4.02

+
+
+
+
+

0.65
1.47
1.08
1.37

Lag time (h)

16.5
23.3
18.5
28.7

+ 4.0
+ 4.6
+ 3.9
_+ 4.2
+

+
+

6.94
5.76
6.03
5.73

+0.55
+0.96
+ 1.66
+ 2.11

24
16
32.7
47.3

+ 4.2
+ 0
+ 7.5
+_ 0.6
**

Spec. resp. inc.

(p-g CO2 g~l
dw soil h - )
0.051
0.062
0.039
0.051

Respiration
delay (h)

__+0.006
+ 0.007
__+0.002
__+0.007
+
+

0
22.5
0
24

0.063 + 0.004
0.053 + 0.001
0.057 + 0.001
0.041 + 0.005

0
0
22.7
44

__+0
-+ 2.5
+ 0
__+1.6

Respiration rate
(~tg CO2 g ~
dw soil h - )
6.60
7.87
7.26
7.00

__+0.40
__+0.15
__+0.48
+ 0.21

6.66
6.11
6.91
7.45

+
+
+
+

***
+
+
+
+

0
0
2.7
0

:,g**

0.27
0.13
0.11
0.27

Statistically significant (ANOVA) effects of tylosin treatment or mercury contamination, heat treatment and the interaction between treatments are
indicated as +, p > 0.1; *, p > 0.05; **, p < 0.01; and * * *, p < 0.001.

Disturbed Microbial Communities

control soil (p = 0.01). Moreover, the soils responded
differently to heat treatment (p -- 0.04), the respiration
rate increasing in the mercury-contaminated soil but
decreasing in the control soil.

Discussion
The colony morphotype data enabled us to detect, in the
case of mercury contamination, disturbance-induced reduction in diversity. Even though these findings are based
upon a subjective determination of morphotypes, investigation of the diversity within morphotypes indicates that
morphotypes are homogeneous [25, 60]. The culturable part
of the microbial community represents only a minor fraction of the total community [2, 15] and only of the dominant
bacteria. The diversity of colony morphotypes thus underestimates the total diversity [1]. Moreover, it is uncertain
whether it is representative of the whole community or even
of the active part of the community [26, 51, 63].
Analysis of the DGGE profiles revealed reduced diversity in both the tylosin-treated and mercury-contaminated
soils. This is in agreement with the findings based on
colony morphology typing in the case of the mercurycontaminated soil, but not for tylosin treatment, where
analysis of the colony morphotype data did not reveal any
differences in diversity. Compared to the colony morphology approach, DGGE may include nonculturable
bacteria. However, as the profiles contain a maximum of
about 50 bands, they clearly do not include all species. It is
important to bear in mind that some bacteria produce
more than one band on the DGGE [41], and that DNA
sequences from different bacteria can have identical
melting properties and hence do not separate on DGGE.
Furthermore, it is doubtful whether extracted and amplified DNA reflects the quantitative abundance of the species
[13, 62]. Whether the bands represent the most abundant
species, the most easily extractable species, the most active
species, or a combination of all these groups is uncertain.
Nevertheless, DGGE seems to be the most sensitive
method for detecting differences in community diversity.
While differences in diversity could be detected from
the colony morphotype and DGGE data, no differences in
diversity could be detected from the substrate utilization
data. The latter reflects a different level of community
structure since the utilization of a specific substrate is not
related to specific bacterial types, as is the case for colonies
and to some extent for DGGE bands. Substrate utilization

55
is attributable to more than one bacterial type [49, 61] and
the degree of substrate utilization is not necessarily
quantitatively related to the n u m b e r of utilizing bacteria
[23]. Because coloration in the analysis depends on bacterial growth, it only describes the potential activity of that
fraction of the microbial community able to grow in the
wells [61[, and not necessarily the potential of the numerically dominant bacteria in the innoculum [49].
We wanted to relate the changes in the diversity of the
microbial community to functional parameters in the soils,
including the ability of the soil system to cope with an additional disturbance (transient heating) that is very different from the initial disturbance (tylosin treatment or
mercury contamination). The heat treatment disturbed the
soil system by changing one or more respiration parameters
in all soils. Furthermore, it must have selected for different
bacteria than tylosin treatment and mercury contamination
since the response of these soils was equivalent to or greater
than that of the respective control soils.
Mercury contamination more strongly affected soil
function than did tylosin treatment. The respiration parameters did not differ significantly between the tylosintreated soil and control soil in the present study, although
inhibition of soil respiration 7 weeks following addition of
tylosin has previously been reported [6]. The effect of heat
treatment seemed to be more or less the same for the
tylosin-treated and control soils, i.e., prolongation of the
lag time and respiration delay, and an increase in the
specific respiration increment.
The most striking differences in the respiration measurements were the enhanced lag time and respiration delay in the mercury-contaminated soil. Furthermore, the
mercury-contaminated soil was more sensitive to the additional disturbance than the control soil since the decomposition of both glucose and alfalfa was inhibited
longer in the contaminated soil than in the control soil
following heat treatment. The lag time [40] or the corresponding decomposition time [24] have been shown to be
sensitive indicators of heavy metal contamination, increasing with increasing contamination. Lag time has been
interpreted as reflecting physiological state, as shown for
E. coil in pure culture studies [36] and for soil microbial
communities [40]. The lag time could also be attributable
to the direct toxic effect of the heavy metals on the microorganisms or in some cases to interactions between the
heavy metal and the involved substrate or enzyme [10].
Also, the size of the microbial biomass could influence the
delay of respiration [40].

56
In the present study, exposure to mercury affected the
respiration delay before commencement of alfalfa decomposition in the same manner but to a greater extent
than the lag time before growth on glucose. This is in
agreement with the finding of Doelman and Haanstra [10]
that lag time is particularly prolonged in the case of material that does not readily decompose. It is possible that
the respiration delay reflects the same thing as the lag
time. However, since the degradation of more complex
substrates is likely to depend on a consortium of microorganisms, this could lead to the interpretation that the
diversity of the microorganisms is important for the duration o f the respiration delay. Prolongation o f the respiration delay could therefore be due to the changes in
diversity, changes in the physiological state of the bacteria,
a direct toxic effect on the bacteria, or a combination of all
factors. It is obvious that other factors than diversity influence the function of the soil community since the
mercury-control soil and the tylosin-control soil differed
in heat tolerance but not in diversity. Instead the difference in stability could be due to, for example, the composition of the community or a lower microbial biomass in
the tylosin-control soil compared to the mercury-control
soil as indicated by the SIR values.
The soils differing most in diversity with respect to
both the culturable and nonculturable part of the microbial population also exhibited the greatest differences in
sensitivity to the additional disturbance. This could be
interpreted as supporting the hypothesis of a link between
diversity and resistance of the ecosystem. The same has
been demonstrated in another soil community, where reduced diversity caused by CHC13 fumigation resulted in
decreased resistance and resilience to additional disturbances [21]. Such an interpretation should be made with
caution, however, as the two disturbances (tylosin treatment and mercury contamination) differ considerably in
terms of duration and target specificity. Thus whereas
tylosin disappears, the organisms are permanently exposed to the mercury. Even though the microbial population adapts to the mercury contamination, the organisms
may expend considerable energy to maintain their tolerance, thereby potentially diminishing their resistance to an
additional disturbance. Since the tylosin disappeared 6
weeks before the measurements of diversity and function
were made, tylosin does not act as a stress factor on the
organisms in the present study. Another explanation for
the reduced resistance of the mercury-contaminated soil is
that organisms selected by the mercury exposure could be

A.K. M/iller et al.

more heat-sensitive than the general population or the
tylosin-resistant population.
Tylosin was chosen to manipulate the soil system in
this experiment because it is specifically toxic to bacteria
and presumably only has secondary effects on other
groups of soil organisms. On the other hand, the effects
of mercury are broader, also affecting the fungi, flora,
and fauna--organisms with important functional roles in
the soil. For instance, mercury had very easily detectable
effects on the vegetation at the site where the soil samples were collected, and soil structure clearly differed
between the soils. The absence of vegetation and resultant changes in substrate levels and microniches in
the soil probably alter the diversity, structure, and
function of the soil community.
The present study thus shows that functional performance was generally unaltered by the transient disturbance, whereas it was slower and more sensitive to an
additional disturbance in the presence of a permanent
disturbance. As regards the relationship between system
diversity and function, we thus found that if diversity was
reduced, system performance was also altered. If there was
no diversity reduction, in contrast, system function was
unaffected. Whether this correlation is attributable to a
relationship between diversity and function or to the use
of different disturbances remains to be elucidated.

Acknowledgments
This research was supported by the "Centre for biological
processes in contaminated soil and sediment" (BIOPRO)
(www.biopro.dk) under The Danish Environmental Research Programme. Special thanks to Dr. Jaap Bloem and
An Vos for their hospitality and assistance with the DGGE,
and to Susanne Eriksen for help with quantification of the
tylosin.

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