FEMS Microbiology Ecology 52 (2005) 139–144
www.fems-microbiology.org
The effects of soil horizons and faunal excrement on
bacterial distribution in an upland grassland soil
Patricia M.C. Bruneau a, Donald A. Davidson b, Ian C. Grieve
Iain M. Young c, Naoise Nunan d,1
b,*
,
a
Scottish Natural Heritage, 2 Anderson Place, Edinburgh, EH6 5NP, Scotland, UK
School of Biological and Environmental Sciences, University of Stirling, Stirling FK9 4LA, Scotland, UK
c
SIMBIOS Centre, University of Abertay Dundee, Bell Street, Dundee DD1 1HG, Scotland, UK
Biomathematics and Statistics Scotland, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland, UK
b
d
Received 24 June 2004; received in revised form 27 October 2004; accepted 29 October 2004
First published online 11 November 2004
Abstract
The density and spatial location of bacteria were investigated within different horizons of an upland grassland soil before and after
a liming treatment to increase the numbers of large soil fauna. Bacterial cells were located by image analysis of stained thin sections
and densities calculated from these data. Excrement from macro- and meso-fauna was identified using micromorphology and the densities of bacteria on specific areas of excrement measured by image analysis. There were significant differences among horizons in the
density of bacterial cells, with the minimum density found in the horizon with least evidence of earthworm activity, but no difference
in density between the organic H and organo-mineral Ah horizons. Soil improvement by liming significantly increased bacterial densities in all three horizons, with the greatest increase found in the horizon with the smallest density before liming.
There were no differences in bacterial density between areas dominated by excrement from earthworms and excrement from
enchytraeids, although densities in both areas were significantly increased by liming. Variability in bacterial density at spatial scales
of less than 1 mm was linked to the occurrence of excrement. Bacterial densities within areas of both types of excrement were significantly greater than those in the surrounding soil. However, the frequency distribution of the ratios of density in excrement to that
in the soil was bimodal, with a majority of occurrences having a ratio near 1 and only some 20–30% having a much larger ratio.
These variations can probably be explained by variations in the age of the excrement and its suitability as a substrate.
2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Soil micromorphology; Soil structure; Biological thin sections; Bacteria; Faunal excrement; Temperate grassland
1. Introduction
The nature of the habitat is of crucial importance in
influencing biological activity within soils [1]. In soil ecology, the concept of functional domains has been used to
*
Corresponding author: Fax: +44 1786 467846.
E-mail address: [email protected] (I.C. Grieve).
1
Present address: CNRS, Biogéochemie des Milieux continentaux,
Bâtiment EGER, aile B, INRA INA-PG, 78850 Thiverval Grignon,
France.
explain the spatial relationships between soil structure
and faunal communities [2,3] and a similar approach
has been successfully applied to biogenic structures
created by the ‘‘engineering’’ activities of meso- and
macro-fauna [4,5]. However, most of the research on
soil–faunal interactions has been concerned with mineral
or cultivated soils and much less is known about the
spatial links between soil structure and fauna in more
organic upland soils. In such soils, fauna have been
shown to play important roles in soil function, most
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsec.2004.10.010
140
P.M.C. Bruneau et al. / FEMS Microbiology Ecology 52 (2005) 139–144
notably in the formation of horizons and structure [6] and
in carbon cycling and organic matter decomposition [7].
Soil micromorphology offers a unique methodology
for studying the nature and spatial distribution of soil
components seen in thin sections [8]. Recent developments in image analysis of soil thin sections [9] offer
exciting possibilities for relating the spatial distribution
of soil microbial populations to specific micromorphological features. Sampling and analysis of bulk soils
have demonstrated variability in microbial activity at
scales of a few mm in the rhizosphere [10] to several meters within a pasture [11]. Micromorphological techniques provide ways of analysing the spatial
distribution of microbial clusters which may be hotspots
of microbial activity and which are likely to be associated with aggregates, particulate organic matter and
the rhizosphere [12]. In one recent study [13], the distribution of bacteria was examined at a range of scales and
bacteria were found to occur in preferentially colonised
patches, close to pores in the subsoil, but more randomly in the topsoil. However, it is not known whether
such spatial patterns also occur within upland soils
which have not been under recent cultivation.
This paper uses micromorphological analysis at two
scales to quantify for the first time the spatial association
between faunal excrement and bacteria in upland soils.
The effects of faunal activity in such soils are most clearly
expressed in the dominant occurrence of excremental features, which varies according to soil horizon and in a variety of structures ranging from distinctive vermiform
features of earthworm origin to amorphous material
resulting from aged or fused excrement. Faunal excrement has been shown to constitute up to 80% of the overall soil volume in the organic horizons and as much as
50% in mineral horizons in upland soils [14]. Excrement
of larger soil fauna provides a habitat for other fauna
and microbes and it is hypothesised here that the spatial
distribution of bacterial colonies will be associated with
excremental features. To test this hypothesis, the spatial
distribution of bacteria was studied by image analysis
of thin sections made from soils from an upland grassland
site. Soils to which a surface application of lime had been
made were compared with unlimed control soils. The aim
of the liming treatment was to stimulate the activity of
soil fauna, particularly earthworms, and thus provide
more substrate material for detrivores. Data on bacterial
distributions derived from soil micromorphology and image analysis were used to test differences in bacterial density between soil horizons and between different types of
excrement in the limed and control soils.
2. Experimental site and methods
Soil samples were obtained from an upland grassland
at Sourhope Research Station, approximately 25 km
south of Kelso in south-eastern Scotland (5528.5 0 N,
214 0 W). The altitude of the site is approximately 300
m, mean annual precipitation is 952 mm [15] and mean
annual soil temperature at 5 cm depth is approximately
8 C. Vegetation is dominated by Agrostis capillaris (L.)
with Festuca ovina (L.), F. rubra (L.) and Anthoxanthum
odoratum (L.). Sheep and cattle were excluded from the
experimental site and plots were mowed and cuttings removed at 3-week intervals during the growing season.
The soil parent material is glacial till derived from
andesitic lavas of Devonian age. Soils are brown forest
soils (Cambisols) belonging to the Sourhope series
[16]. The upper part of the soil profile typically comprises thin L and F horizons (total thickness 1–4 cm),
a 3–8 cm thick H horizon and an Ah horizon with total
thickness of between 10 and 20 cm. Some profiles exhibited a thin (1–1.5 cm) dark reddish grey to dark grey
(5YR 4/2 to 10YR 4/1) horizon at the base of the H horizon. Microscopic study of thin sections from this horizon revealed that it was organic, but contained a high
concentration of phytolith fragments and this horizon
was designated Hphy [14].
This paper examines differences in bacterial distributions between the control plots and plots which received
a surface dressing of calcium carbonate (39% Ca) at 0.6
kg m2 in the spring of 1999, 2000 and 2001. The annual
application of calcium carbonate increased the mean pH
of the organic (H) horizon from 3.4 to 4.1 between 1999
and 2001 [14]. Two years after the first liming treatment
was applied, the limed plots had significantly larger
abundance and biomass of earthworms than the unlimed control plots [17]. When each species was analysed
separately, there were significantly more individuals of
Dendrobaena rubidus and Aporrectodea rosea in the
limed plots. One year after the first application of lime,
there was a significant reduction in the total number of
enchytraeid worms, but no reduction in total biomass.
Cognettia was replaced by Fridericia as the dominant
genus [18]. These changes in fauna following liming were
also reflected in the excrement visible on thin sections
and the liming treatment increased the amount of earthworm excrement [14]. Earthworm excrement was mainly
found in the upper, organic horizons and that of enchytraeids was dominant in the organo-mineral horizon
(Ah). Despite significant heterogeneity, soil improvement treatments also influenced the structure and functioning of the bacterial community, with liming having
the greatest impact. Autotrophic ammonia oxidation
was significantly increased by the combined action of
liming and a sewage sludge application [19].
Undisturbed soil samples were taken from the organic
and upper Ah horizon using cores (6 cm diameter by 5
cm length). Two replicates samples were obtained from
each plot in June 1999, November 1999 and September
2001. Bacteria were fixed as soon as possible after sampling with an aqueous solution of glutaraldehyde and
P.M.C. Bruneau et al. / FEMS Microbiology Ecology 52 (2005) 139–144
subsequently stained with an aqueous solution of Calcofluor white MR [9]. Thin sections were prepared using
standard water/acetone exchange in the liquid phase
and impregnated using Crystic XB52n9 resin. Vertically
oriented, diamond-polished thin sections, 30 lm thick,
were produced from the impregnated blocks.
Fig. 1 summarises the methodology by which faunal
excrement and bacterial locations and density statistics
were determined. At the macroscale each thin section
was divided into homogeneous areas which corresponded to the principal soil horizons. It has already
been shown that there are two distinctive types of faecal material in this upland grassland soil [6]. Small
pedofeatures (60–100 lm), spherical to ellipsoidal in
shape, loose to moderately compacted, mainly organic
and with no internal fragments larger than 20 lm were
derived primarily from enchytraeids. Larger excremental pedofeatures, up to several millimetres in size, often
with mammillated shapes and including mineral and
plant fragments and with diagnostic vermiform features, were derived from earthworms [8]. These morphological features were identified using AnalySIS
v3.0 (Soft-Imaging System GmbH, Munster, Germany)
with an analysis procedure specifically designed for this
project [20].
To identify bacterial clusters, thin sections were
examined using a Zeiss Axioplan 2 microscope fitted
for epifluorescence (100W OSRAM mercury UV lamp,
HBO 103 W/2) at a magnification of ·630. The slides
were mounted on a motorised stage and tessellated
images of 25 (5 · 5) contiguous fields of view, representing an area of 0.282 mm2, were acquired within each defined horizon. Bacterial locations were determined using
141
a Zeiss KS300 Imaging System 3.0 [9]. The x and y coordinates of each individual bacterial cell and its shape
and size were determined. Total bacterial counts in each
·630 image were then converted to bacterial densities
(mm2). These densities can be related to soil volumes
on the assumption that the thickness of the analysed
soils in which bacteria could be seen was 2 lm (±1 lm
from the plane of observation). A measured density of
1000 cells mm2 is thus equivalent to a density of
5 · 108 cells cm3.
In order to compare the locations of bacteria and faunal excrement, bacterial locations were converted to a
new reference system at a ·50 scale of magnification
and overlain on the image of excremental features.
One consequence of this scale change was that bacterial
locations merged where distances apart were less than
the new pixel size, although bacterial colonies in soils
consist of very small numbers of cells [21]. Each ·50 image was segmented into areas of similar excremental features and bacterial density (mm2) was calculated for
each defined area of excrement. These densities could
not be compared directly with densities calculated at
the ·630 magnification scale due to the effects of the
change of scale on bacterial clusters. Densities were
therefore calculated for each whole image at the ·50
magnification scale; the two density measures were very
strongly correlated (r = 0.953; P < 0.001). Total void
space and void space per area of interest were measured
by applying a manual threshold to the ·50 image. A
threshold value was chosen for each image to separate
void space from dark material. Bacterial densities on
each type of excrement were then corrected for void
space.
Fig. 1. Identification of bacteria and excremental features in soil thin sections. (a) Single image at magnification ·50, identification of areas with
similar pedofeatures; (b) tessellated images (5 · 5) at magnification ·630, identification of bacteria; (c) Details showing a bacteria cluster.
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P.M.C. Bruneau et al. / FEMS Microbiology Ecology 52 (2005) 139–144
3. Results and discussion
3.1. The effect of horizons and a liming treatment on
bacterial density
Table 1 shows bacterial densities in each horizon for
the control and limed soils and striking contrasts are
suggested. Mean densities ranged from under 200 to
over 1300 cells mm2, equivalent to 108 and 6.5 · 108
cells cm3, respectively. These densities are similar to
densities measured by other methods for upland soils.
Densities of bacterial colony forming units (cfu) of
4 · 1012 cfu m2 in the uppermost 5 cm of improved
grassland soils have been reported for a range of sites
in the UK uplands [22], equivalent to just less than 108
cfu cm3.
Frequency distributions of the raw bacterial densities
showed slight positive skewness and the data were normally distributed following a square root transformation. Statistical analyses were carried out on the
transformed dataset and all tables report back-transformed means and 95% confidence limits. Two-way
analysis of variance showed that both the treatment effect (P < 0.001) and the horizon effect (P < 0.05) were
statistically significant, but the interaction between
treatment and horizon was not significant. Mean bacterial density was lowest for the Hphy horizon of the unlimed soil, probably reflecting the limited current
faunal activity in this horizon. The phytoliths which
were a prominent feature of the micromorphology of
the Hphy horizon were highly fragmented and this horizon contained a large amount of older undifferentiated
excrement, the result of substantial past bioturbation
[14]. Microbial activity and diversity are smaller in deeper soil organic horizons that contain older organic matter [23] as a result of the rapid decrease in microbial
activity and biomass with age of the substrate [24].
The only micromorphological evidence for earthworm
activity in the Hphy horizon at Sourhope was the existence of vertical channels, suggesting that this horizon
may be primarily a zone of transit for earthworms between the upper and lower horizons, as opposed to their
main habitats.
Table 1
Means and 95% confidence limits of density of bacterial cells (backtransformations of square root-transformed data) in three horizons of
the Sourhope soil
Horizon
H
Hphy
Ah
Control
Limed
Mean density
(mm2)
95% CL
Mean density
(mm2)
95% CL
944
183
804
654–1287
42–423
559–1094
1275
1085
1316
1004–1579
728–1516
1016–1654
Bacterial densities in the H and Ah horizons were not
significantly different, and the structure of both these
horizons contained distinctive faunal excrement. Earthworm excrement was most clearly expressed in the H
horizon and excrement of enchytraeids was the dominant form observed in the Ah horizon [14]. It would thus
appear that both organisms provide substrates to sustain bacterial activity within these horizons.
Bacterial densities were greater in all three horizons
of the limed soil and this treatment effect was highly significant (P < 0.001). This is consistent with previous
studies which have reported increased bacterial activity
and diversity, community respiration and dissolved organic carbon leaching following liming of acidic forest
soils [25]. In a range of upland grassland soils in the
UK [22], liming increased bacterial numbers, bacterial
activity and carbon utilisation, although the almost 4fold differences in bacterial numbers between improved
and unimproved grassland soils in that study were much
larger than the differences in bacterial densities with liming found here.
3.2. Associations between bacteria and faunal excrement
Table 2 shows the mean densities of bacteria on areas
of excrement from: (a) enchytraeids and (b) earthworms,
again as back-transformations of statistics calculated on
square root-transformed data. Densities were calculated
on the basis of the areas of mineral and organic matter
excluding void space. Liming effects were highly significant (P < 0.001) for bacterial density on both types of
excrement, with significantly greater densities in the
limed soils. Horizon effects were also significant
(P < 0.01 for enchytraeid excrement and P < 0.05 for
earthworm excrement). The smallest densities were
again found on excrement in the Hphy horizon, and there
was no significant difference between the H and Ah horizons or earthworm and enchytraeid excrement.
Bacterial densities on each type of excrement were
also compared with densities in the soil. For every sampling area, the density of cells on the excrement and
within the whole sampling area was calculated. Differences between the densities were normally distributed
and, for both excrement types, a paired t-test showed
significantly greater densities (P < 0.01) on the areas of
excrement than in the whole sampling area. The mean
difference for earthworm excrement was 207 (standard
error = 66) cells mm2 and that for enchytraeid excrement was 141 (standard error = 47) cells mm2.
Fig. 2 shows frequency distributions of the ratio of
density on excrement to density in soil for: (a) enchytraeid and (b) earthworm excrement. The distributions were bimodal in both cases, with most excrement
densities similar to soil densities (ratios of near 1) but
between 20% and 30% of excrement densities substantially greater than in the soil. These greater densities
P.M.C. Bruneau et al. / FEMS Microbiology Ecology 52 (2005) 139–144
Table 2
Means and 95% confidence limits of density of bacterial cells on: (a)
enchytraeid and (b) earthworm excrement (back-transformations of
square root-transformed data) in three horizons of the Sourhope soil
Horizon
Control
Limed
95% CL
Mean density
(mm2)
95% CL
(a) Enchytraeid excrement
H
778
91
Hphy
Ah
710
533–1070
43–157
500–966
1481
805
1373
1175–1822
486–1206
1046–1744
(b) Earthworm excrement
H
951
212
Hphy
Ah
656
591–1397
67–438
385–997
1306
914
1481
1016–1633
585–1318
1105–1914
Mean density
(mm2)
143
H and Hphy horizons, formed by coalescence and ageing
of excrement [14]. The existence of high densities of bacteria linked to the spatial distribution of faunal excrement in the structure of these soils provides further
support for the spatial association of hotspots of microbial activity with labile substrates [12]. The analysis of
stained soil thin sections has also revealed substantial
variations in microbial abundance at a much more detailed spatial scale than previously seen [10,11]. The
application of similar analytical methods to cultivated
soils revealed a spatial association between bacteria
and pores [13]; this study demonstrates a further link
with faunal excrement in an uncultivated soil.
4. Conclusions
(a) Enchytraeid
70
60
Frequency
50
40
30
20
10
0
0
1
2
3
4
5
Ratio
(b) Earthworms
Frequency
30
20
10
0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Ratio
Fig. 2. Frequency distributions of the ratio of bacterial density on
excrement to that in whole soils for: (a) enchytraeid and (b) earthworm
excrement.
may be on areas of relatively fresh excrement as suggested by the distinct clusters of bacteria within excremental features in Fig. 1(c). Microbial biomass and
activity on earthworm excrement decrease rapidly with
substrate age [24]. Large amounts of undifferentiated
excrement occur in these soils, particularly in the lower
In this upland grassland soil, the upper horizons are
dominated by organic matter consisting largely of excrement. Although there are many difficulties in associating
excremental features with particular organisms, earthworms and enchytraeids seem to have been the main
sources. Densities of bacteria, determined by counting
under high magnification on stained thin sections of soil,
were similar to those determined by other methods. Bacterial densities were similar in the organic H horizon
and the uppermost organo-mineral Ah horizon in the
horizon, dominated, respectively, by earthworm and
enchytraeid excrement. Bacterial densities were significantly less in an intervening horizon rich in phytolith
fragments and which had the smallest concentration of
earthworm excrement, presumably a consequence of
the unpalatable phytolith fragments. The effect of liming
was to increase densities significantly in all three upper
horizons, with the most marked change in the phytolith-rich horizon. The changes in the earthworm and
enchytraeid populations stimulated by liming were
followed by increased amounts of faunal excrement,
an attractive substrate for bacteria [14].
There was no significant difference in density between
sites on earthworm and enchytraeid excrement,
although mean densities on both substrates were significantly increased by liming and were significantly greater
than densities in the whole soil. The major implication
of the data presented here, however, is the importance
of fresh faunal excrement in determining the distribution of high concentrations of bacteria at spatial scales
of less than 1 mm. These data suggest than spatial
variability in microbial function exists at the sub-mm
level within the spatial structure of upland soils.
Acknowledgements
This work was funded by a NERC Grant (GST/02/
2127) under the Soil Biodiversity and Ecosystem
144
P.M.C. Bruneau et al. / FEMS Microbiology Ecology 52 (2005) 139–144
Function thematic programme. We thank David Crabb,
Kirsty Harris and Dr. Linda Deeks (Soil-Plant Dynamics
Unit, Scottish Crop Research Institute) for preparation
and analysis of the thin sections for biological analysis
and George MacLeod (Stirling University) for preparation of thin sections for micromorphological analysis.
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