Influence of earthworm invasion on soil

Soil Biology & Biochemistry 34 (2002) 1929–1937
www.elsevier.com/locate/soilbio

Influence of earthworm invasion on soil microbial biomass
and activity in a northern hardwood forest
Xuyong Lia, Melany C. Fiskb,*, Timothy J. Faheyb, Patrick J. Bohlenc
a

Institute of Geography Science and Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China
b
Department of Natural Resources, Cornell University, Ithaca, NY 14853, USA
c
Archbold Biological Station, Lake Placid, FL 33852, USA
Received 26 June 2001; received in revised form 11 September 2002; accepted 19 September 2002

Abstract
Recent invasion and activity of exotic earthworms has profoundly altered the chemical and physical environment of surface soils in
northern hardwood forests that previously had mor humus horizons. We investigated the influence of earthworm invasion on soil microbial
biomass and activity in surface soils of Allegheny northern hardwood forests in central New York state. Earthworm activity in these sites had
transformed surface soils with clear Oi, Oe, and Oa horizons (forest floor) overlying mineral soil, to more uniformly mixed organic-enriched
A horizons. The highest concentrations of microbial biomass and activity occurred in the forest floor. Microbial biomass (assayed by

chloroform fumigation – extraction) nearly doubled in surface (0 – 5 cm) mineral soils in response to earthworm activity, an effect that
corresponded directly to redistribution of organic matter from forest floor into the mineral soil. Microbial activity in surface mineral soils was
even more sensitive to the presence of earthworms than microbial biomass. For example, substrate-induced respiration (or maximum initial
respiratory rate, MIRR) was 6.7-fold greater, basal respiration was 5-fold greater, and microbial respiration per unit microbial biomass
(metabolic quotient, qCO2) was almost 3-fold greater in surface mineral soils where earthworms were present than in earthworm-free sites.
Of the activity indices, only MIRR was higher when expressed on an organic matter basis. Surface mineral soils where earthworms were
present thus appear to retain a high proportion of the microbial biomass and activity found in mor organic horizons. Our findings suggest that
earthworm activity stimulates the activity of soil microorganisms, probably by enhancing organic C availability via processing and mixing of
litter. The relative pattern in microbial properties did not change over the growing season; however, there were some seasonal changes in the
proportional differences between worm and no-worm soils. Our results indicate interactions among earthworms, organic matter, and soil
microbial activity that should alter the carbon and nutrient balance of northern hardwood forest surface soils, relative to non-invaded soils.
q 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Earthworm invasion; Microbial biomass; Microbial activity; Maximum initial respiration response; Metabolic quotient; Northern hardwood forest;
Soil organic matter

1. Introduction
The soil microbial community drives nutrient transformations and thus plays a major role in nutrient cycling of
ecosystems. Under suitable environmental conditions, the
extent of the turnover of organic compounds will be mainly
controlled by both the quantity and activity of the microbial

biomass (Martens, 1995). A better understanding of the
dynamics of soil microbial biomass and activity and their
responses to natural and human-caused disturbances is
* Corresponding author. Address: Department of Biology, Appalachian
State University, Boone, NC 28608, USA.
E-mail address: fiskmc@appstate.edu (M.C. Fisk).

essential to improving our ability to predict patterns of
nutrient cycling in ecosystems and their response to natural
or human-induced disturbances.
In the north temperate and boreal forests of North
America, native earthworms are rare due to eradication by
Pleistocene glaciations (Gates, 1976; Reynolds, 1995), but
active invasion by exotic earthworms is occurring in many
regions, including the hardwood forests of the northeastern
United States (Burtelow et al., 1998). The dominant exotic
earthworms in this region are European species (Lumbricus
rubellus, an epi-endogeic species; L. terrestris, and anecic
species) and Asian species (Amynthas hawayanus, an epiendogeic species). Earthworms play a major role in altering
development of the soil profile, especially near the soil

0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
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X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

surface (Edwards and Bohlen, 1996). The invasion by exotic
earthworms has been linked to significant changes in
organic matter breakdown and nutrient dynamics
(Langmaid, 1964; Scheu and Parkinson, 1994; Alban and
Berry, 1994; Bohlen and Edwards, 1995; Steinberg et al.,
1996; Scheu, 1997; Burtelow et al., 1998). These changes
can be expected to have significant effects on soil microbial
biomass and activity. However, in forest ecosystems, the
effect of exotic earthworm invasion on soil microbial
biomass and activity is not well understood.
The objective of the present study was to quantify the
effect of earthworm invasion on the quantity and activity of

microbial biomass in a northern hardwood forest ecosystem.
We employed the standard technique of chloroform
fumigation and extraction (FE) as a measure of microbial
biomass (Vance et al., 1987) together with the microbial
metabolic quotient (qCO2) (Anderson and Domsch, 1990)
and maximum initial respiratory response (MIRR) (Beck
et al., 1997) as indices of microbial activity. We hoped to
demonstrate how earthworm invasions interact with spatial
and temporal environmental variation to affect both
microbial populations and their metabolic activity in forest
soils. These studies can then help us to interpret related
responses of soil nutrient availability and soil solution
chemistry to earthworm invasion of northern hardwood
ecosystems.

2. Materials and methods
2.1. Study sites
The study sites were located at Arnot Forest in central
New York, situated in the northern Allegheny Plateau
physiographic province. (428160 N, 768280 W). Annual rainfall is 100 cm and average summer and winter temperature

are 22.0 8C and 2 4.0 8C, respectively. Soils are acidic
Dystrochrepts with a well-developed organic horizon
averaging about 4 cm thick and overlying an acidic (pH
4.5 –5.0) mineral horizon. Soils in the study area are well
drained and exhibit pit and mound microtopography. In
each of three separate sites, paired sample plots
(20 m £ 20 m) were established in adjacent locations that
had either been invaded recently by exotic earthworms
(worm plot) or still remained worm-free (no-worm plot).
Worm and no-worm plots in two sites (Site 1 and Site 2)
were dominated by sugar maple (Acer saccharum ), basswood (Tilia americana ), and white ash (Fraxinus americana ). Worm and no-worm plots in the third site (Site 3)
were dominated by aspen (Populus tremuloides ) and oak
(Quercus rubra ). Further information about forest composition and dynamics at Arnot Forest is available (Fain et al.,
1994; Fahey, 1998).
Earthworm populations were assessed in all plots in April
1999 and June 2000 using a standard formalin extraction
technique (Raw, 1959). Eight liters of 0.25% formalin was

applied to four (1999) or five (2000) 0.25 m2 areas within
each plot and earthworms emerging from the soil were

collected into vials containing 4% formalin and returned to
the lab for identification. Samples were taken from plots
designated as earthworm plots as well as from plots
designated as worm-free reference plots, but the very few
number of individuals collected from worm-free plots were
negligible.
2.2. Soil sampling
Our aim was to quantify effects of the presence of
earthworms on microbial biomass and activity, and to test
whether those effects are consistent seasonally. In mid-July
we intensively sampled all three sites. To examine seasonal
patterns we sampled in Site 1 on four dates through the
snow-free season (May 17, July 17, September 5 and
November 8, 2000). Each sample collection consisted of
four replicate soil cores (5 cm diameter) collected at random
locations in each plot.
Our sampling was designed to compare the surface
horizons where biological activity is concentrated and
earthworm effects are most noticeable. In no-worm plots,
each soil core was separated into forest floor (average

4.7 cm depth) and 0– 5 cm mineral soil (NW FF and NW 0–
5, respectively). These were typical mor profiles and the
forest floor consisted of well-developed Oe and Oa (humus)
horizons beneath the fresh litter (Oi). The forest floor had
been eliminated in the worm plots; no Oe or Oa horizons
remained and the Oi was present until about mid-summer.
The surface horizon in the worm plots was an organicenriched mineral (A). Each soil core in the worm plots was
separated into 0– 5 and 5 – 10 cm mineral soil (W 0– 5 and
W 5 –10, respectively).
Samples were homogenized by hand, taking care to
minimize disruption of soil structure. Coarse fragments
(. 2 mm) and fine roots were removed. Samples were
refrigerated at 2 – 4 8C for approximately 24 h before
processing and incubations.
2.3. Sample analyses
Soil organic matter content was estimated from the ashfree dry mass (450 8C, 4 h). Microbial biomass C was
determined using the chloroform fumigation– extraction
(FE) procedure (Brookes et al., 1985; Vance et al., 1987).
For each sample, one subsample was extracted with 0.5 M
K2SO4 in a ratio of 1:10 (w/v) for forest floor samples and

1:5 (wt:vol) for mineral soil samples. A second subsample
was fumigated with chloroform for 5 days in a vacuum
desiccator, followed by extraction in 0.5 M K2SO4.
Subsamples of extracts were sealed in glass ampules for
oxidation of DOC (Menzel and Vaccaro, 1964) and CO2 in
the ampule headspace was analyzed on a CO2 coulometer
(Huffman, 1977). Microbial biomass C was estimated as the
difference in extractable DOC between fumigated and

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X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

unfumigated soils using a correction factor (Kc) of 0.45
(Vance et al., 1987).
Microbial respiration and the maximum initial respiratory rate (MIRR; Martens, 1995; Beck et al., 1997) also were
assayed using the substrate-induced respiration (SIR)
approach (Anderson and Domsch, 1978). For each sample,
two replicate subsamples (fresh weight equivalent of 5 g dry
weight for forest floor, 10 g dry weight for mineral soil)

were incubated in air-tight jars at 22 8C. Following a 12 h
pre-incubation, one replicate of each sample received
glucose solution at a rate of 10 mg C per g soil for the
surface organic-rich horizons (no-worm forest floor and
worm mineral 0 –5 cm), or 5 mg C per g soil for the
subsurface mineral horizons (no-worm 0 – 5 cm, worm 5 –
10 cm), in 3 ml deionized water. Preliminary incubations
showed that these were optimum C concentrations for
stimulating a maximum respiratory response over the first
6 –8 h of incubation. The second replicate received an
equivalent amount of deionized water (3 ml). Water
addition had a proportionately larger effect on water content
of mineral soils than forest floor, which had higher water
content to begin with.
Jar headspace was sampled at 0 and 5 h and CO2
concentrations were analyzed by gas chromatography.
Basal respiration rates were estimated as the rate of CO2
accumulation in jars receiving deionized water only; MIRR
was estimated as the rate of CO2 accumulation in jars
receiving glucose. Microbial qCO2 was determined by

dividing basal respiration (BR) (mg CO2 – C per g dry soil
h21) by FE microbial biomass C (g Cmic per g dry soil)
(Anderson and Domsch, 1990).
2.4. Statistical analyses
Statistical analyses were performed using SPSS for
Windows Release 10.00. Two-way ANOVA (GLM

multivariate) was used to test effects of earthworm presence
(comparing 0 –5 cm mineral soil in worm and no-worm
plots) and sites for mid-July data, or earthworm presence
and sampling dates for seasonal data in Site 1. The microbial
biomass and activity indices were calculated and tested on
both a unit soil dry mass and organic matter (OM) basis.
Differences among means of specific treatments and
different soil horizons were tested with Student—Newman – Keuls method. We also used SPSS to analyze the
correlations between FE microbial biomass, MIRR, qCO2
and organic matter content, soil moisture, and average
monthly soil temperature.

3. Results

Earthworms were absent from our no-worm plots, and
averaged 158 individuals/m2 in worm plots at the time of
our surveys (Table 1). Forest floor (Oi, Oe, and Oa horizons)
depth averaged 4.7 cm in no-worm plots. Earthworm
invasion had mixed the surface organic and mineral
horizons, eliminating the Oe and Oa and resulting in a
surface (0 – 5 cm) mineral soil horizon that was significantly
enriched in organic matter (Table 2). Mineral soil water
content also was higher in the presence of earthworms
(Tables 2 and 3).
Microbial biomass and activity exhibited significant
effects of earthworm presence for the 0 –5 cm mineral soil
horizon in mid-July (Table 4). With the exception of
microbial biomass, differences caused by site location in 0–
5 cm mineral soils were not significant, and there were no
significant interactions between earthworm presence and
site. Soil microbial biomass and activity also differed
significantly between worm and no-worm soils in the
seasonal analysis (Table 5). Sampling date was significant
for BR and for the maximum initial respiratory response

Table 1
Earthworm population density and biomass estimated from samples collected from forest plots with worms in April 1999 and June 2000. Data are not presented
for no-worm plots because abundance was negligible (,3 individuals/m2). Standard errors of the mean are in parentheses, n ¼ 3. There were no significant
differences between sample dates in number or biomass of any earthworm species or all species combined (P . 0.05). The ecological category of the
earthworm provides our best estimate of the feeding behavior of the different earthworm species at our site, recognizing that there is overlap between categories
under different ecological conditions and at different life stages
Species

L. rubellus
Lumbricus terrestris
Octolasion tyrteum
Aporrectodea tuberuculata
Dendrobaena rubida
Eisenia rosea
Lumbricus immatures
Total
a

Ecological category/feeding behaviora

Epi-endogeic
Anecic
Epi-endogeic
Endogeic
Epigeic
Epigeic
N/A

Population density (no./m2)

Biomass (g/m2)

1999

2000

1999

2000

67 (26.2)
10 (9.7)
59 (29.5)
4 (3.8)
0.3 (0.3)
2 (2.3)
7 (7.3)
150 (29.2)

85 (38.4)
4 (3.9)
54 (30.1)
14 (8.7)
1 (1.3)
0
6 (5.5)
165 (40.8)

22 (7.4)
9 (8.9)
10 (4.6)
2 (1.9)
0.02 (0.02)
0.4 (0.4)
0.4 (0.4)
43 (2.5)

28 (11.9)
8 (8.3)
7 (3.4)
4 (2.2)
0.3 (0.3)
0
1 (1.1)
48 (3.1)

Anecic: vertical burrowing species that inhabits mineral soil and feeds on litter at the soil surface; endogeic: horizontal burrowing species that is active in
both the organic and mineral soil; epigeic: surface dwelling species feeding on surface organic matter; epi-endogiec: species intermediate in behavior between
epigeic and endogeic species.

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X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

Table 2
Organic matter content (%) and gravimetric soil water content (%; dry
weight basis) for soils sampled in mid-July, 2000. Incubation moisture was
calculated based on the quantity of water added to soils prior to incubations.
Standard errors of the mean are in parentheses, n ¼ 3 sites
Organic
matter
No-worm
Forest floor
Mineral soil,
0–5 cm
Worm
Mineral soil,
0–5 cm
Mineral soil,
5–10 cm

Field
moisture

Incubation
moisture

40 (8.7)
9 (0.7)

92 (28.0)
35 (1.4)

152
65

22 (1.7)

63 (1.9)

93

9 (0.4)

40 (2.1)

70

Table 4
Results (F-values and significance) of ANOVA testing effects of treatment
(earthworms present or absent) and different sites on microbial biomass and
activity in the surface mineral soil horizon (0–5 cm depth) of Arnot Forest
soils (n ¼ 24)
Dependent variablea

FE
FE/OM
MIRR
MIRR/OM
BR
BR/OM
qCO2

Source
Worm treatment

Site

Treatment £ site

8.70**
7.15*
28.86***
9.20**
49.85***
7.72*
11.36**

3.61*
0.70
1.30
0.85
0.65
0.78
0.66

1.04
0.29
1.57
1.13
3.03
1.62
0.81

*P , 0.05, **P , 0.01, ***P , 0.001.
FE: FE microbial biomass C per gram dry soil basis; FE/OM: microbial
biomass C per gram organic matter basis; MIRR: MIRR respiration rate per
gram dry soil basis; MIRR: MIRR respiration rate per gram organic matter
basis; qCO2: qCO2 value with FE microbial biomass C basis.
a

(MIRR), but not for FE biomass (Table 5). Significant
interactions in the seasonal analysis were found only for BR
(Table 5).
Microbial biomass concentrations in surface horizons
were significantly lower in the presence of worms (W 0– 5)
than in their absence (NW FF). Comparison of all soil
horizons using the Newman – Keuls test showed that the
means of FE microbial biomass decreased in the order: NW
FF . W 0 –5 . NW 0 –5 ¼ W 5 – 10. Means of FE/OM
differed between surface and subsurface horizons, in the
order: NW 0 – 5 ¼ W 5 –10 . NW FF ¼ W 0– 5 (Fig. 1);
hence, the differences in FE-biomass between NW FF and
W 0 – 5 appear to be associated primarily with the
redistribution of soil organic matter by worms. Seasonal
variation in FE microbial biomass was minimal for all but
NW FF, which peaked in September (Fig. 2).
BR rates per g soil in mid-July were similarly higher in
NW FF than W 0– 5, and followed the same overall pattern
among soil horizons as FE biomass (Fig. 3). This was true
also for the respiratory potential of soil microorganisms per
Table 3
Gravimetric soil water content (%; dry weight basis) in soils collected from
Site 1 in May, July, September, and November. Standard errors of the mean
are in parentheses, n ¼ 4 samples
17 May

17 July

Field moisture (%)
No-worm
Forest floor
85 (2.9) 67 (12.0)
Mineral soil, 0–5 cm
41 (2.8) 32 (2.6)
Worm
Mineral soil, 0–5 cm
56 (6.8) 64 (1.6)
Mineral soil, 5–10 cm 36 (3.1) 38 (2.1)
Incubation moisture (%)
No-worm
Forest floor
145
127
Mineral soil, 0–5 cm
71
62
Worm
Mineral soil, 0–5 cm
86
94
Mineral soil, 5–10 cm
66
68

5
September

9
November

58 (6.2)
30 (2.5)

82 (14.2)
47 (4.2)

48 (3.5)
34 (2.8)

73 (5.5)
37 (1.9)

118
60

142
77

78
64

103
67

g soil (MIRR), which in the surface mineral soil (0 – 5 cm)
was almost 7 times higher in worm than no-worm plots in
mid-July (Fig. 4). In fact, the worm effect was observed
even at greater depths, as MIRR per g soil at 5 –10 cm depth
in worm plots was significantly higher than that of 0 – 5 cm
depth in no-worm plots (Fig. 4). Basal respiration on an
organic matter basis was more variable and did not differ
among horizons (Fig. 3), suggesting that much of the
earthworm effect on microbial respiratory activity was
associated with redistribution of organic matter. MIRR was
slightly more sensitive to earthworm effects, however, and
MIRR/OM in W 0– 5 cm was intermediate to NW FF and
NW 0– 5 (Fig. 4).
Both BR and MIRR exhibited the same relative pattern
among soil horizons throughout the growing season (Fig. 2)
but, unlike FE-biomass, differed significantly among
sampling dates (Table 5). MIRR increased overall in the
autumn and the proportional difference between worm and
no-worm 0 –5 cm mineral soils was slightly greater in May
and July than in the autumn (Fig. 2). Basal respiration was
notably high in worm relative to no-worm 0 –5 cm mineral
soils in May and July, but not September or November.
The metabolic quotient (qCO2) followed the same trend
Table 5
Results of ANOVA (F-values and significance) testing effects of treatment
(earthworms present or absent) and sampling date on the microbial biomass
and activity in the surface mineral soil horizon (0–5 cm depth) of Arnot
Forest soils (n ¼ 32)
Dependent variable

FE
MIRR
BR
qCO2

Source
Worm treatment

Date

Treatment £ date

22.14***
90.00***
44.07***
9.74**

1.79
28.39***
4.17*
1.54

0.69
0.17
5.36**
2.87

X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

1933

Fig. 1. Chloroform fumigation extraction (FE) microbial biomass in worm
and no-worm soils in three sites, July 2000. Bars are standard errors of the
mean, n ¼ 12. Different letters indicate significant differences according to
the Student—Newman– Keuls method, P , 0.05. NW FF: forest floor of
no-worm plots; NW 0– 5: 0–5 cm mineral soil horizon of no-worm plots;
W 0 –5: 0–5 cm mineral soil horizon of worm plots; W 5–10: 5–10 cm
mineral soil horizon of worm plots.

as FE and MIRR, but surface horizons did not differ
significantly: NW FF ¼ W 0 –5 . NW 0 –5 ¼ W 5 –10
(Fig. 5). In the surface mineral soil the presence of
earthworms increased qCO2 by almost 3-fold in midsummer. Seasonally, however, this effect was observed only
in May and July and no differences were found in September
and November (Fig. 2).
Earthworm invasion markedly increased organic matter
as well as soil moisture content in the surface mineral soil
(Tables 2 and 3), and these changes probably affected
microbial biomass, MIRR and qCO2 significantly. Overall,
these environmental factors were more strongly correlated
with the activity than with the size of the soil microbial
biomass. For example, the correlation coefficient between
field soil moisture and MIRR (R ¼ 0.73, P , 0.001) was
greater than that between field soil moisture and FE
microbial biomass (R ¼ 0.60, P , 0.001). Similarly, a
slightly stronger correlation was observed between soil
organic matter and MIRR (R ¼ 0.90, P , 0.001), than
between soil organic matter and FE microbial biomass
(R ¼ 0.77, P , 0.001). No relationships between microbial
biomass or activity and seasonal variation in soil temperature were detected.

4. Discussion
When earthworms invade acidic forest soils with a mor
humus layer, over a period of a few years they can mix the
organic horizons and the mineral soil by their feeding
activity (Langmaid, 1964; Alban and Berry, 1994). Earth-

Fig. 2. Seasonal patterns of FE microbial biomass C, BR, MIRR, and qCO2
in worm and no-worm soils of Site 1. Bars are standard errors of the mean,
n ¼ 4 samples.

worms vary in their feeding behaviors and hence the specific
manner in which they impact the soil profile and organic
matter distribution. In our study sites, the dominant species
(L. rubellus and O tyrteum ) are epi-endogeic, and are active
in and process organic matter in both organic and surface
mineral soils. The anecic L terrestris, the next most
abundant species, likely transports deeper mineral soils to
the surface and feeds on surface organic matter. Consistent
with these ecological categories of earthworms, we
observed a surface layer A horizon in earthworm plots
that was intermediate in organic matter content to the forest
floor and underlying mineral soil in reference, no-worm
plots. Both surface and deeper-dwelling earthworms rapidly

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X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

Fig. 5. Metabolic quotient in worm and no-worm soils in three sites, July
2000. Bars are standard errors of the mean, n ¼ 12. Different letters indicate
significant differences according to the Student—Newman–Keuls method,
P , 0.05. See Fig. 1 for explanation of legends.

Fig. 3. Basal respiration in worm and no-worm soils in three sites, July
2000. Bars are standard errors of the mean, n ¼ 12. Different letters indicate
significant differences according to the Student—Newman–Keuls method,
P , 0.05. See Fig. 1 for explanation of legends.

process fresh litterfall and mineral soil is exposed at
the surface during summer and early autumn, accelerating
the decomposition and mineralization of litter. Earthworms
also affect soil structure by forming burrows and casts, and
they produce organic compounds (Lee, 1985) that become
concentrated in their casts and along burrow walls (Edwards
and Bohlen, 1996; Tiunov and Scheu, 1999). Through these
mechanisms of organic matter redistribution and processing, earthworms directly and indirectly affect the composition, abundance and activity of soil microorganisms.
We observed a significant increase in FE microbial
biomass in the surface mineral soil layer (0 –5 cm) of a

Fig. 4. MIRR in worm and no-worm soils in three sites, July 2000. Bars are
standard errors of the mean, n ¼ 12. Different letters indicate significant
differences according to the Student—Newman– Keuls method, P , 0.05.
See Fig. 1 for explanation of legends.

northern hardwood forest ecosystem in response to
the invasion of exotic earthworms, principally L. rubellus,
L. terrestris, and Octolasion tyrtaeum. The increase in FEbiomass in worm plot surface mineral soils probably was
associated with the increase in organic matter concentration
of this soil layer, because FE/OM was similar between the
surface horizons (worm 0 – 5 cm and no-worm forest floor).
However, FE/OM actually was reduced in the 0 – 5 cm
worm soils compared to 0– 5 no-worm soils, suggesting that
microbial biomass did not increase to the same extent as
organic matter following earthworm invasion.
Other studies of earthworm effects on microbial biomass
in forests have reported inconsistent responses. Burtelow
et al. (1998) observed increased microbial biomass in
temperate deciduous forest in New England, whereas
reductions were reported in beech in Europe (Wolters and
Joergensen, 1992) and pine forest in southwestern Alberta
(McLean and Parkinson, 1997). The increase in FE
microbial biomass per unit organic matter reported by
Burtelow et al. (1998) contrasts with our results and
indicates a variable influence of exotic earthworms that
may depend on soils, forest or worm species, and possibly
length of time since invasion. Burtelow et al. (1998) studied
Asian species (Amynthas ), in contrast to the European
Lumbricus and Octolasion spp. common in our sites. The
annual life cycle and epi-endogeic feeding behavior of
Amynthas may distinguish its effects from those of the
perrenial, anecic and epi-endogeic species common at our
sites. Furthermore, experimental work in mesocosms has
shown that differential effects of earthworms on microbial
biomass and activity depend not only on the ecological
categories of earthworms present, but also on the species
composition within those categories (Scheu et al., 2002).
Scheu and Parkinson (1994) observed that European
earthworms of another genus (Dendrobaena ) caused a
decrease in microbial biomass in the L/F layers but an
increase in the H and Ah horizons of aspen forests. In their
study, introduction of earthworms had changed the soil
profile to a lesser extent, probably because earthworm
introduction was more recent and possibly because the
epigeic Dendrobaena carries out less mixing between
organic and mineral horizons. Nevertheless, these results

X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

concur with ours and suggest that soil microbial biomass is
shifted deeper down in the soil profile as a result of
earthworm redistribution of surface soil organic matter.
For example, in our study FE microbial biomass was as high
in the 5– 10 cm soil depth in worm-invaded plots as in the
0 –5 cm depth in the worm-free sites.
Microbial activity was enhanced in the presence of
earthworms to a greater extent than could be explained by
changes in organic matter content alone. Barley and
Jennings (1959) first reported that earthworms increased
soil respiration, and similar results have been reported by
Ross and Cairns (1982), Haimi and Huhta (1990) and
Ruz-Jerez et al. (1992). Our results reinforce and extend
these findings. In particular, we observed in the surface
mineral soil of the worm-invaded plots an increase in the
maximum initial respiratory response (MIRR), expressed on
both a soil dry mass and an organic matter basis, and in the
microbial metabolic quotient (qCO2). The MIRR is largely
determined by the respiration rate of soil microorganisms
that are stimulated by glucose addition and is intended as an
index of the active soil microorganisms (Wardle and
Parkinson, 1990), contrasting with the FE which includes
the biomass of dormant soil microorganisms. The qCO2,
calculated as the ratio of basal microbial respiration/FE
biomass, provides an index of the overall activity of the total
microbial pool. The increases in MIRR/OM and qCO2 in the
worm-invaded surface mineral soil suggest that earthworms
enhance resource availability or ameliorate environmental
conditions that limit the activity of the soil microflora in
worm-free mineral soils.
The most likely explanation of this earthworm effect on
microbial activity is the mixing of labile organic substrates
into the upper mineral soil horizons. In support of this
supposition is the observation that both MIRR/OM and
qCO2 are highest in the forest floor horizon and intermediate
in the upper mineral soil of the worm-invaded plots and
lowest in the mineral soil of the no-worm plots. We interpret
this pattern as an indication that the overall quality of
organic substrates is highest in the forest floor where fresh
litter and fine root exudation and turnover provide a
continuous supply of labile substrates for microbial
metabolism. Conversely, much of the organic matter in
the upper mineral soil of the no-worm plots is probably
condensed, recalcitrant humic substances, not readily
available to most soil heterotrophs. The upper mineral soil
of the worm plots would contain a mixture of the organic
matter categories. Metabolism of that organic matter also
may differ due to the influence of earthworm mixing and
consumption on fungal communities (McLean and Parkinson, 2000).
Other effects of the earthworms on both soil resources
and environmental conditions also could contribute to the
patterns we observed. Earthworms produce labile substrates
as secretion and excretion products (Lee, 1985), and watersoluble, low-molecular-weight compounds are assimilated
by the rapidly multiplying microbial community in the

1935

earthworm gut (Barois and Lavelle, 1986). Also, Binet et al.
(1998) have proposed that microbial metabolism is
stimulated by low concentrations of chemical mediators
released by earthworms. The effects of earthworm activity
on soil structure could improve environmental conditions
such as moisture and aeration to favor microbial activity.
Although we have no direct evidence for these possible
effects, we did observe moderately high linear correlations
between soil water content and both MIRR/OM (R ¼ 0.51)
and qCO2 (R ¼ 0.52). The moisture content of the 0 –5 cm
mineral soil was consistently higher in the worm-invaded
plots than the no-worm plots, probably as a result of forest
floor interception of summer rains and possibly increased
water-holding capacity associated with higher organic
matter content in mineral soil of the worm-invaded plots.
As a consequence of the insulating effects of the forest floor,
soil temperature of the surface mineral soil was slightly
lower during summer in no-worm plots (0.5 – 1 8C), whereas
this pattern was reversed during the autumn. However, these
differences did not correspond to patterns of microbial
properties when incubated at uniform temperature in the
laboratory.
Microbial responses to the presence of earthworms
should depend on the stage of invasion, and our results
suggest that microbial activity remains elevated beyond the
initial colonization period. Langmaid (1964) documented
the complete transformation of surface organic horizons of
acidic podzols to well-mixed mineral soils in 3 years
following earthworm invasion. The initial consumption of
organic layers when earthworms first invade can support
high populations that peak in as little as 4 years and decline
somewhat thereafter (Ligthart and Peek, 1997), and may
also cause a transient stimulation of microbial activity. For
example, studies early in colonization history suggest that
mineralization of soil organic matter is enhanced (Alban
and Berry, 1994; McLean and Parkinson, 1997) as microbial
processes respond to the dramatic changes in organic matter
availability. Following this initial pulse of organic matter
metabolism, microbial activity might be expected to
stabilize over time in response to earthworm modifications
of annual carbon inputs and the longer-term soil physical
environment. In aspen-hardwood forests small changes in
soil organic content may continue, but the initial pulse of
mineralization appears to have occurred in the first 6 years
following invasion (Alban and Berry, 1994). However, the
corresponding changes in microbial activity have not been
documented. We do not know when exotic earthworms
colonized our sites or whether the organic matter content of
the soil has reached a new lower equilibrium level following
an initial pulse of mineralization that may have
occurred in the early stages of invasion. However, soil
organic C in worm plots was 25% lower than in no-worm
plots (P. J. Bohlen, unpublished data), indicating that a
significant loss of organic matter has already taken place in
invaded plots. Our results thus indicate a continuation of
enhanced microbial activity in mineral soils, probably

1936

X. Li et al. / Soil Biology & Biochemistry 34 (2002) 1929–1937

associated with environment and with earthworm effects on
availability of annual organic matter inputs.
These effects on decomposer microorganisms are likely a
combination of direct organism interactions and indirect
effects due to transformations of the physical and chemical
soil environment. This study is intended to demonstrate
general differences in microbial activity in forest soils with
and without earthworms, and we cannot separate these
influences. Nevertheless, seasonal patterns do give some
insight into direct and indirect effects. Seasonal analysis in
Site 1 showed consistency over time in the relative patterns
between treatments and among soil horizons; hence, the
results reported here for mid-July probably are generally
applicable throughout the growing season. The general
consistency over time in microbial activity suggests an
indirect influence of earthworms via soil organic matter
distribution and availability and soil physical properties.
Direct comparisons of earthworm and microbial activity
over time are needed to verify this explanation. Subtle
seasonal changes did suggest some differences in the timing
of microbial activity between worm and no-worm plots. For
instance, seasonal patterns of qCO2 and BR suggest that the
earthworm effect on the activity of microbial biomass is
greater in spring and summer than in autumn, possibly as a
result of earthworm mixing of the previous year’s litter early
in the season. The seasonal pattern of microbial activity also
differed between no-worm forest floor and worm 0 – 5
mineral soil. Our indices of microbial activity (BR, MIRR,
qCO2) were similar between forest floor and worm surface
mineral soils in July, but differed in the autumn months
(Fig. 2). These patterns are suggestive of direct effects of
earthworms on microbial activity; again, data comparing
earthworm to microbial activity are needed to test this idea.
In conclusion, we have demonstrated significantly
greater microbial biomass and activity in surface mineral
soils where earthworms are present. It is important to
interpret this change in microbial activity in surface mineral
horizons in the context of a changed soil profile that no
longer has a surface forest floor horizon. Although organic
matter concentration of surface mineral soils approached
that of no-worm forest floor, slightly lower microbial
activity (indexed by MIRR/OM and qCO2) suggested that
mixing of organic matter into the mineral soil matrix has
changed its availability for microbial utilization. The
quantitative effect of this change in the nature of organic
matter depends on changes in total soil pools. Nevertheless,
it appears that the transport and change in availability of
organic matter has the potential to affect nutrient availability, especially if microbial immobilization of nutrients is
inherently greater in the organic-rich forest floor. Concentrations of inorganic P were higher in soil leachate in these
worm plots (D. Pelletier 2001, MS Thesis, Cornell
University), and labile P pools also were higher in surface
mineral horizons (E. Suarez 2001, MS Thesis, Cornell
University). These differences in availability of mineral
nutrients may be related to the microbial responses to

earthworms in surface soils of these forests. Changes in the
timing of microbial activity that appear to result from
earthworm activity also may affect nutrient pools via
changes in the timing of nutrient mineralization relative to
that of plant nutrient uptake.

Acknowledgements
We thank Suzanne Wapner, Dana Briel, Kurt Smemo,
Derek Pelletier, Maryann Welsch, Noel Gurwick and Beth
Lawrence for help in the field and laboratory. This work
would not have been possible without the project support
and valuable discussions provided by Peter Groffman. This
research was supported by a grant from the National Science
Foundation of USA.

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