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Soil Biology & Biochemistry 32 (2000) 2055±2062
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Changes in microbial biomass C, N, and P and enzyme activities in soil
incubated with the earthworms Metaphire guillelmi or Eisenia fetida
Bao-Gui Zhang*, Gui-Tong Li, Tian-Shou Shen, Jian-Kui Wang, Zhao Sun
College of Agricultural Resources and Environment, China Agricultural University, Beijing 100094 People's Republic of China
Accepted 6 June 2000
Abstract
Earthworms are ubiquitous in soil and ingest large amounts of soil, organic matter and leaf litter. To assess changes in organic matter
fractions after passage through the earthworm gut, we measured microbial biomass C, N and P and the fungal-to-bacterial ratios in wormworked soil (WWS), obtained by incubating soil for 24 h with large numbers of the anecic earthworm Metaphire guillelmi (1:5 ratio of fresh
weight worms: dry weight soil). Microbial biomass C, N and P were estimated by the fumigation±extraction methods, and fungal-to-bacterial
ratios by selective inhibition using substrate induced respiration. Enzyme activities in the gut of M. guillelmi were also compared with an
epigeic earthworm species Eisenia fetida. Activities of cellulase, protease, chitinase, acid and alkaline phosphatases, in gut, casts and
uningested soil were measured.
In WWS, microbial biomass decreased (130 mg C g 21 soil), and there was a concomitant increase of available nutrients (27 and 10 mg g 21
soil for ninhydrin-reactive N and inorganic P, respectively). There was no difference between the glucose-sensitive microbial biomass (MB)
and the control but the respiratory quotient was greater (2.85 ^ 0.17 and 2.95 ^ 0.07 mg CO2-C g 21 soil h 21 for WWS and control,
respectively). The fungal-to-bacterial ratio was slightly higher in WWS than in uningested soil (1.61 vs. 1.35).
Cellulase activity was greater in the gut of the epigeic earthworm than in that of the anecic one (152.8 ^ 18.7 vs. 18.9 ^ 1.3 mg
glucose g 21 worm fw h 21); conversely, protease and phosphatase activities were signi®cantly higher in gut of the anecic specie as opposed
to the epigeic species. The activity of cellulolytic enzymes was slightly higher in casts than in soil; while activities of protease, acid (pH 6.5)
and alkaline (pH 9.0) phosphatases were lower in earthworm casts than in the uningested soils (protease, acid and alkaline phosphatase
activity were 29.5 ^ 1.7 mg tyrosine g 21 worm fw h 21, 570 ^ 2.9, 748 ^ 7.3 mg p-nitrophenol g 21 soil h 21 in soil and 17.8 ^ 2.0 mg
tyrosine g 21 worm fw h 21, 327 ^ 26.7, 549 ^ 19.7 mg p-nitrophenol g 21 soil h 21 in casts, respectively). We conclude that micro-organisms
are used by earthworms as a secondary food resource, and that passage through earthworm gut decreases the total soil MB and increase the
active components of MB. q 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Earthworm; Microbial biomass; Protease; Phosphatase; Cellulolytic enzyme; Substrate-induced respiration
1. Introduction
Earthworms are ubiquitous soil animals and dominate the
invertebrate biomass in temperate soils. Worms ingest a
large amount of soil, organic matter and surface litter. In
soils where they are abundant, the surface layers may be
considered as earthworm casts of different age (Lavelle,
1978). Thus earthworms are thought to have a profound
in¯uence on soil organic matter decomposition and nutrient
transformations. Earthworms prefer a diet of decomposed
and decomposing organic matter, which suggests that
micro-organisms are also ingested as food (Edwards and
Fletcher, 1988). As micro-organisms are a key factor in
soil nutrient transformations and the soil microbial biomass
* Corresponding author.
E-mail address: zhangbg@mail.cau.edu.cn (B.-G. Zhang).
(MB) serves both as a pool and as a source of plant nutrients,
ingestion of MB by worms could affect these processes and
nutrient supply. Previous work in this area has given contradictory ®ndings. Plate counts of proteolytic bacteria, total
bacteria and actinomycetes were reported to be increased by
passage through earthworm intestinal tracts (Parle, 1963;
Daniel and Anderson, 1992; Devliegher and Verstraete
1995); while Pedersen and Hendriksen (1993) reported
that numbers of some bacteria increased and others
decreased. MB was reported to decrease (Bohlen and
Edwards, 1995; Devliegher and Verstraete 1995), increase
(Scheu, 1992) or remain unchanged (Daniel and Anderson,
1992) after passage through the earthworm gut.
Little information is available concerning the capacity of
earthworms to degrade organic matter. Most studies on
earthworm digestive enzymes have concentrated on
Lumbricidae (Zhang et al., 1993), with cellulases being
0038-0717/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00111-5
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B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
the most commonly studied enzymes (Parle, 1963; Loquet
and Vinceslas, 1987; UrbaÂsÏek, 1990). Proteases, phosphatases and chitinases, important in soil N and P transformations, have been less well studied in earthworms.
Our objectives were, ®rstly, to determine the effects of
passage through earthworm guts on the soil microbial
community, especially microbial biomass C, N, P and
respiration, and secondly, to determine the activity of the
digestive enzymes of earthworms and their casts.
2. Materials and methods
2.1. Soil and earthworms
Surface soil (0±10 cm) was taken from the experimental
station in the campus of China Agricultural University,
Beijing. The soil had the following physical and chemical
properties: pH (in H2O) 7.4, clay 16%, silt 41%, sand 43%,
soil organic matter 1.8%, total N 0.1%. The soil was airdried and sieved (,2 mm) and well mixed. Four parts (v/v)
of soil were mixed with one part of sieved (,2 mm) wheat
straw compost; the mixture was adjusted to 50% water holding capacity and were conditioned at 258C at least 10 d
before the experiment began.
Specimens of the anecic earthworms Metaphire guillelmi
(Sims and Easton, 1972) were collected by digging from a
green food production base in a Western suburb of Beijing
and cultivated in the laboratory for 1 month. Adult earthworms, with fresh weight varies between 2.3 and 2.5 g, were
washed free from soil and blotted with ®lter paper before
fresh weight determinations were made. The earthworms
were placed in the incubated mixture for 1 d prior to the
start of the experiment, in order to replace their gut contents
by the mixture. Adult epigeic earthworms Eisenia fetida (fw
varied from 0.4 to 0.5 g) were purchased from the Chinese
Agricultural Academy's laboratory stock.
2.2. Microbial biomass determination
To ensure the soil contained a large proportion of earthworm casts and that the casts in the worm-worked soil
(WWS) could be relatively homogeneous in age, a large
number of earthworms were added to a comparatively
small volume of soil and a short treatment time was chosen.
The gut transit time varies from 2 to 20 h for most species
(Parle, 1963; Lavelle, 1978; Lee, 1985), so a residence time
of 24 h was selected for the experiment. A preliminary
experiment showed that by adding 1 g fresh weight worms
to every 5 g dry weight soil, more than 50% of soil passes
through earthworm intestine during 24 h of treatment.
Although the earthworms were a little crowded, there was
no weight loss during the experiments.
To a sample of 200 g (dw equivalent) of the conditioned
soil, earthworms were added at a ratio of 1 g fresh weight
worms to every 5 g (dw equivalent) soil. After 24 h at 258C,
the earthworms were withdrawn. The worm-worked soil
was further incubated for 3 d (3 d WWS) at 258C with the
soil moisture adjusted to 50% water holding capacity. The
worms removed from the pots were washed free from soil,
weighed and reintroduced into another sample of the conditioned soil. After 24 h, the earthworms were removed, the
WWS were further incubated for 2 d (2 d WWS). The earthworms were washed once more, weighed and re-introduced
into a new sample in order to obtain 1 d WWS. Then, each
WWS sample was homogenised and subsampled in triplicate to determine soil moisture content and measure microbial biomass C, N and P. Worm-free soils were used as the
controls and were treated in the same way as the WWS
samples.
For biomass C and N estimation, triplicate samples of
15 g (dw equivalent) were extracted with 80 ml of 0.5 M
K2SO4 immediately after exposure to worms or after a
subsequent 24 h fumigation with CHCl3. Organic C in the
extracts was measured in 5 ml aliquots by the dichromate
oxidation method (Vance et al., 1987). Ninhydrin-reactive
N (Nnin) in the extracts was analysed by reacting aliquots
(2 ml) with a ninhydrin reagent (Moore and Stein, 1954) and
reading absorbance at 570 nm. Microbial biomass C and N
were calculated, respectively, by the equations Bc EC £
2:64 (Vance et al., 1987) and Bn EN £ 5:0 (Joergensen
and Brookes, 1990), where EC and EN are the difference
between organic C or ninhydrin-reactive N extracted by
0.5 M K2SO4 from fumigated and non-fumigated soil,
respectively.
For microbial biomass P determination, triplicate samples
of 15 g were extracted with 80 ml of 0.5 M NaHCO3 (pH
8.5) immediately at the end of incubation or after a subsequent 24 h fumigation with CHCl3. Inorganic P (Pi) was
analysed in aliquots (1 ml) of the extracts by the ammonium
molybdate±ascorbic acid method described by Murphy and
Riley (1962). A spike of KH2PO4 equivalent to 25 mg P g 21
soil was used to correct for Pi ®xation during the NaHCO3
extraction. Biomass P was calculated by Bp EP =0:38;
where EP is the difference between NaHCO3-Pi extracted
from fumigated and non-fumigated soil (Brookes et al.,
1982). NH41-N and NO32 were measured in the same extracts
as microbial biomass C and N. NH41-N was analysed by
indophenol blue method (Keeney and Nelson, 1982),
NO32-N was analysed by a colorimetric method of nitration
of salicylic acid (Cataldo et al., 1975).
2.3. Substrate-induced respiration (SIR) rate and bacterialto-fungal ratio
Triplicate WWS samples were obtained by exposing
earthworms to conditioned soil for 24 h, at a 1:5 ratio of
worms to soil (fw/dw). Then, SIR was determined in triplicate samples (ca. 10 g dry weight) of WWS or uningested
soil with a continuous air-¯ow system modi®ed and adapted
from Cheng and Coleman (1989). Glucose was added to soil
with talcum as a carrier. Preliminary experiments showed
that 10 mg glucose g 21 soil was optimal for a maximal
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
response. Streptomycin and cycloheximide (Sigma Chemical Co. St Louis, MO) were used as inhibitors of bacterial
and fungal respiration respectively, the inhibitors were used
separately and also combined. A preliminary experiment
showed that 8 mg g 21 was the optimal concentration for
inhibition of respiration, and a greater streptomycin concentration stimulated soil respiration. As streptomycin contains
N, and non-target micro-organisms (e.g. fungi) may use it as
a source of N, in samples not amended with streptomycin, N
was added as (NH4)2SO4 solution allowing all treatments to
have the same quantity of N. The mean respiratory rates of
0.5±2.5 h after addition of glucose and inhibitor(s) were
used to calculate SIR and to evaluate the effects of the
inhibitors. Selectivity of inhibitors was estimated by the
additivity ratio (expressed as sum of reduction in
substrate-induced respiration with separate addition of
streptomycin and cycloheximide divided by reduction
caused by the application of both inhibitors in combination).
The metabolic quotient of the soil MB qCO2 was obtained
by dividing basal respiration rate by total soil MB and
expressed as CO2 evolved per unit microbial C per unit
time (Anderson and Domsch, 1978).
2057
phenol reagent (Lowry et al., 1951) was added and the
mixture was homogenised immediately. Absorbance at
700 nm was determined 20 min later and related to those
of tyrosine standards (0±1000 mM) treated in a similar way.
Acid (pH 6.5) phosphatase activity was determined
according to the method adapted from Tabatabai (1982).
Centrifuged homogenate (500 ml) solution was incubated
with 4 ml of modi®ed universal buffer (MUB) at pH 6.5,
and 1 ml of 25 mM sodium p-nitrophenyl phosphate in pH
6.5 MUB at 378C. After 30 min, the reaction was stopped by
adding 4 ml of 0.5 M NaOH solution. After a further 20 min
at room temperature, the concentration of p-nitrophenol was
determined by reading the absorbance at 420 nm and related
to those of 1% p-nitrophenol standards.
Chitinase activity was determined by incubating 1 ml of
centrifuged homogenate with 1 ml of citrate-phosphate
buffer pH 5.2 and 1 ml of 0.2% chitin suspension for 24 h
(Skujins et al., 1965). After stopping the reaction in boiling
water and centrifugation, liberated b-N-acetyl-d-glucosamine in the supernatant was measured as described by Reissig et al. (1955).
2.5. Enzymatic activity in casts and soil
2.4. Enzymatic activity in gut of the earthworms
Adult earthworms were washed free from soil and incubated in soil or soil/decomposed straw mixture (worm to
soil or mixture at 1:5 ratio, fw/dw) for 24 h. Sub-samples
of fresh casts were collected, the earthworms were washed
and weighed. Four individuals of M. guillelmi were sampled
and dissected. After dissection, the gut wall and its contents
of one M. guillelmi worm were homogenised in a known
volume of 50 mM pH 4.8 citrate buffer at 48C to determine
cellulase activity, or in pH 8.1 Tris±HCl buffer to determine
protease activity. The homogenates were centrifuged for
10 min at 6000 rpm, the supernatant was stored at 2188C
until the enzymes were assayed. Because of their small size,
E. fetida (2 individuals for each replication, 6 replications)
were homogenised without dissection. The enzymatic activities of both species were expressed per unit fresh weight of
worm.
Cellulase activity was determined using a ®lter paper
assay (Mandels et al., 1976). Brie¯y, 1 ml of 50 mM pH
4.8 citrate buffer, 1 ml centrifuged homogenate, 0.5 ml
toluene and a ®lter paper strip were incubated at 508C for
24 h. The reducing-sugar concentration was estimated by
the dinitrosalicylic acid method (Miller, 1969). Glucose
was used as the standard for reducing-sugars.
Protease activity was measured as described by Ladd and
Butler (1972). Centrifuged homogenate (1 ml) was incubated with 2.5 ml of 1% sodium caseinate in 100 mM Tris
buffer pH 8.1 at 508C. After 60 min the reaction was stopped
by precipitating the enzyme and the remaining caseinate by
adding 1 ml of 17.5% trichloroacetic acid. After centrifugation, 2 ml of the supernatant was mixed with 3 ml of 1.4 M
Na2CO3. Then 30 min later, 1 ml of 2-fold diluted Folin-
Protease activity of earthworm casts and soil was determined using the same method as for earthworm extracts.
Preliminary experiments showed that no glucose liberated
from degradation of cellulose or ®lter paper under conditions of our test was detectable by the DNS method (Miller,
1969) or by reducing sugars methods (Smoggyi, 1945).
Thus the CO2 evolution rate from casts and soil incubated
with micro-crystalline cellulose (MCC) was used as an indicator of cellulolytic enzyme activity. Earthworm casts or
soil (20 g) were mixed thoroughly with 200 mg MCC, and
incubated at 258C for 7 d. CO2 evolved was absorbed by
5 ml of 1.0 M NaOH which was replaced daily. Residual
alkali was titrated against 300 mM HCl after precipitating
carbonate by excess of BaCl2, with phenolphthalein as indicator.
Acid (pH 6.5) and alkaline (pH 9.0) phosphatase activities of casts and soil were determined by the method
described by Tabatabai (1982).
Enzymatic activities of earthworms are presented on a
live weight basis, and other results are presented on an
oven-dry soil weight basis. All results are the means of
independent assays (number of mean are indicated in corresponding tables and ®gures). Student's t-test was used to
compare signi®cant difference between the means.
3. Results
3.1. Effect of earthworm on total soil microbial biomass
Soil microbial biomass (MB), C, N and P had decreased
after 24 h of earthworm treatment, while soil extractable
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B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Table 1
Soil microbial biomass, soluble organic C and available N and P for each treatment. Standard errors of the mean n 3 are shown in parenthesis
Treatment
Control
Earthworm treatment
Microbial biomass a (mg g 21 soil)
Soil extractable C, N and P (mg g 21 soil)
C
N
P
Dichromate-oxidable organic C
Ninhydrin-reactive N
NaHCO3-inorganic P
920.6 (67.5)
791.1 (11.1)
*b
205.25 (3.4)
146.35 (10.9)
***
102.9 (6.6)
74.9 (1.7)
**
84.6 (7.6)
110.7 (7.9)
*
10.43 (0.02)
37.54 (0.29)
***
94.3 (1.6)
104.1 (1.6)
**
a
Kc for biomass C, N, P were used as 0.41, 0.2 and 0.37, respectively.
*, ** and *** represent, respectively, P , 0:05; P , 0:01 and P , 0:001 probability of signi®cant difference between earthworm treatment and control (ttest).
b
organic C, ninhydrin-reactive N (Nnin) and NaHCO3-Pi had
increased (Table 1).
In the WWS, Nmin consisted mainly of NH41-N, which was
nitri®ed during the subsequent incubation as indicated by an
increase of soil NO32-N (Fig. 1). Inorganic P was higher in
WWS than in the control soil and the amounts increased
during the subsequent incubation (Fig. 2).
combined inhibition reached 41% for the earthworm treatment and 48% for the control. Inhibitor selectivity was satisfactory, as the inhibitor additivity ratio was close to 1.0
(1.12 and 1.04 for control and WWS, respectively).
3.2. Effect of earthworms on the soil microbial community
Cellulase activity was 7-fold greater in the guts of E.
fetida than that in the guts of M. guillelmi (Table 2). In
contrast, protease and acid phosphatase activity of E. fetida
were signi®cantly lower than in M. guillelmi
(135.5 ^ 1.2 mg tyrosine g 21 fw h 21, 474 ^ 0.11 mg pnitrophenol g 21 fw h 21) (Table 2).
Chitinase activity was not detected in the gut of either of
the two earthworm species.
Although the total MB decreased during the earthworm
treatment, the glucose-sensitive component of the MB was
not considerably affected, as there was no signi®cant difference in glucose-induced respiration between the earthworm
treatment and the control (2.85 ^ 0.17 and 2.95 ^ 0.07 mg
CO2-C g 21 soil h 21 for WWS and control, respectively).
The Metabolic quotient (qCO2), was signi®cantly P ,
0:05 higher in WWS than in the control soil (10.66 ^ 1.9
and 5.09 ^ 0.48 mg CO2-C mg 21 microbial biomass-C h 21
for WWS and control, respectively). This resulted from a
combination of an increase of basal respiration and a decline
of MB in the earthworm treatment. The fungal-to-bacterial
ratio of the glucose-sensitive MB was slightly higher in
WWS than in the uningested soil (1.61 vs. 1.35). The total
Fig. 1. Effects of earthworm on soil ninhydrin N and mineral N. Bars
represent standard errors.
3.3. Cellulase, protease and phosphatase activities in
earthworm gut
3.4. Digestive enzymes in soil and casts of Metaphire
guillelmi
Activities of protease, acid and alkaline phosphatases in
the casts of the earthworm M. guillelmi were 17.8 ^ 2.0 mg
tyrosine g 21 soil h 21, 327 ^ 26.7 and 549 ^ 19.7 mg pnitrophenol g 21 soil h 21, respectively, while those in the
Fig. 2. Effect of earthworm on soil inorganic P. Bars represent standard
errors.
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
2059
Table 2
Activity of digestive enzymes in gut of two earthworm species, means and standard errors and numbers of the mean (in parenthesis)
Enzymatic activity a
Earthworm
E. fetida
M. guillelmi
a
b
Cellulase
Protease
Phosphatase
152.8 18:7; n 6
18.9 1:3; n 4
*** b
24.6 0:3; n 4
135.5 1:2; n 4
****
127.0 0:11; n 3
473.9 4:9; n 3
**
Expressed respectively as mg glucose, mg tyrosine, and mg p-nitrophenol produced g 21 earthworm live weight h 21.
**, ***, ****: signi®cantly different between E. fetida and M. guillelmi at P , 0:001; P , 0:0001 and P , 0:00001; respectively (t-test).
control soil were 29.5 ^ 1.7 mg tyrosine g 21 soil h 21,
570 ^ 3 and 748 ^ 7.3 mg p-nitrophenol g 21 soil h 21,
respectively (Table 3).
The CO2 evolution rates were higher in casts than in the
control soil when a mixture of soil and decomposed wheat
straw was used as the medium for earthworm culture, which
suggested that the microbial activity was greater in casts
compared to the control soil (Fig. 3). The difference of the
CO2 evolution rate between worm casts and soil was maximum at day 5, with the CO2 evolution rate of the casts being
3-fold greater than that in soil. Cellulose amendment did not
further increase CO2 evolution rate either in earthworm
casts or in the control soils.
When the earthworm culture medium was soil (without
wheat straw addition), in the absence of cellulose, there was
no difference of CO2 evolution rate between casts and uningested soils, except on day 2, where there was a slight
increase in casts; however, cellulose amendment caused a
higher CO2 evolution rate in earthworm casts compared to
the uningested soils (Fig. 4).
4. Discussion
4.1. Effects of earthworm on micro-organisms
Our data and published information shows that various
components of soil MB respond differently to passage
through earthworm gut. This partly explains why different
results were obtained depending upon which components of
the biomass were measured. Total soil MB is assessed by
Table 3
Activity of digestive enzymes in soil and in fresh casts of Metaphire guillelmi Means n 4 and standard errors of the mean (in parenthesis)
Phosphatase activities (mg pnitrophenol g 21 soil h 21)
Treatments
Soil
Casts
a
Acid
570 (2.9)**
327 (27)
Protease
activity (mg
tyrosine g 21
soil h 21)
Alkaline
a
748 (7.3)***
549 (19.7)
29.5 (1.7)**
17.8 (2.0)
**, ***: signi®cantly different between soil and casts at P , 0:01 and
P , 0:001; respectively (t-test).
chloroform-fumigation methods, SIR method measures the
active MB or more exactly the substrate sensitive component of MB, while plate counting methods provide a
measure of the number of cultivable (or viable) microorganisms. In our study, passage through the earthworm
gut reduced total soil MB (Table 1), which is in accordance
with results reported by Devliegher and Verstraete (1995)
and Bohlen and Edwards (1995). These authors also used
fumigation technique to determine MB. However, Daniel
and Anderson (1992) found no change of soil MB after
gut passage although they used the same fumigation-extraction method. In their experiment, the comparison of MB was
made between earthworm casts and uningested soil; but that
may be an effect of the selective behaviour of earthworms
on soil MB. Earthworms selectively consume soil fractions
with high concentration of micro-organisms (Hendriksen,
1990), and not all the ingested micro-organisms are killed
during passage through earthworm intestine (Edwards and
Fletcher, 1988; Hendriksen, 1990). In that study, the detrimental effect of the earthworm gut on soil MB would have
been counteracted by enriching casts with micro-organisms
through selective feeding behaviour (Hendriksen, 1990). In
our study, soil MB was compared between WWS and the
uningested soils, thus the indirect effect of earthworm selective feeding was eliminated.
In WWS, a reduction in soil MB with a concomitant
increase of extractable organic C, mineral N and
NaHCO3-extractable Pi indicated that release of microbially
immobilised nutrients was enhanced by earthworms (Table
1). The increase in metabolic quotient qCO2 of soil MB in
the WWS suggested a rejuvenation of the microbial community, as the qCO2 quotients of ªyoungº micro-organisms are
frequently greater than that of ªagedº ones (Anderson and
Domsch, 1978). The release of immobilised nutrients may
have a stimulating effect on microbial activity and activate
dormant microbes. Fischer et al. (1997) demonstrated that
passage of microbes through earthworm guts resulted in a
signi®cant increase in the number of vegetative cells and a
decrease in the number of spores of Bacillus megaterium.
Their results suggested that germination of bacterial spores
were enhanced by the gut passage.
In summary, soil total MB was negatively affected by
passage through earthworms gut while active MB (glucose
sensitive MB) increased. Release of microbially-immobilised
2060
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Fig. 3. CO2 evolution rate of soil and casts. Soil was amended with a wheat
straw residue (soil/residue ratio 4:1, w/w) and incubated for 24 h with or
without earthworms (soil:worm ratio 5 dw/fw) without (A) or with (B)
cellulose amendment (avicel, 1% w/w).
nutrients, rejuvenation of the microbial community and
possibly activation of dormant microbes may have
enhanced the microbial activity in the earthworm casts.
Reduction of soil MB by the earthworm M. guillelmi
indicates that this earthworm may consume soil microorganisms. Compared to the epigeic earthworm E. fetida
which feeds on plant litter, the gut of M. guillelmi has
much greater protease and phosphatases activities and
much lower cellulolytic enzyme activities (Table 3). This
suggests that M. guillelmi may assimilate proteins and
organic P compounds in the cells of soil micro-organisms
rather than the cellulose of plant litter. The preference for
partially decomposed organic materials rich in micro-organisms rather than for fresh organic residues by earthworms
(Cortez et al., 1989; Moody et al., 1995) is also an indication
that earthworms may consume micro-organisms. In the
course of organic residue decomposition, exhaustion of
more easily decomposable constituents such as sugar and
proteins precedes that of cellulose and hemicellulose.
Substances remaining in partially decomposed plant residues are mainly cellulose, hemicellulose, lignin, substances
newly synthesised by micro-organisms and microbial cells.
Lignins are very resistant to microbial attack, and microbial
metabolites are thought to be more recalcitrant than the
initial substances. Since cellulase activities were very
weak in the guts of M. guillelmi, microbial biomass must
be the only substrate for the earthworms. Thus it is easy to
speculate that plant residues are used as primary resources
by micro-organisms which in turn, are used by M. guillelmi
as a secondary resource. As for E. fetida, the relatively
higher cellulase activity in the gut may allow them to utilise
plant materials directly as a food source. The results of the
enzymatic activities in the gut and casts of M. guillelmi
suggested that proteins and phosphorus-containing organic
compounds are digested in the gut rather than in the casts,
whilst cellulose seems to mainly be degraded in casts.
Consumption of micro-organisms by earthworms may
have various consequences on organic matter decomposition. Release of microbial immobilised nutrients and rejuvenation of soil MB may enhance the decomposition of soil
organic matter as metabolic activities of micro-organisms
remain intense. When earthworm feeding intensity is high
and soil nutrients are limited, earthworm feeding may have
detrimental effects on microbial population and activity,
consequently the decomposition rate of organic residues
may be reduced. Although the soil fungal-to-bacterial
ratio was not signi®cantly modi®ed by earthworms in our
experiment, there is other evidence (Pedersen and Hendriksen, 1993; Toyata and Kimura, 1994) that the composition
and succession of the microbial community may be changed
by earthworms. Earthworm preferentially feed on fungi
degrading water-soluble sugars and cellulose, while the
lignin-degrading fungi characteristic of the later stage of
plant litter decomposition were less selected (Moody et
al., 1995). It is well known that the capacity to decompose
complex organic matter varies with microbial community
(Campbell, 1983).
4.2. Digestive enzymes in gut and casts of the earthworms
Fig. 4. CO2 evolution rate of soil. Soil was incubated for 24 h with (- - -) or
without (±) earthworms M. guillelmi (soil:worm ratio 5 dw/fw), with (1)
or without (2) cellulose amendment (avicel, 1% w/w).
The difference in the enzymatic activities of the two
earthworms studied may be associated with their ecological
categories and feeding habitats. The earthworm E. fetida is
an epigeic species which feeds mainly on plant litter. Their
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
high cellulase activity allows them to ef®ciently assimilate
plant materials, which are composed mainly of cellulose;
while M. guillelmi is an anecic species which feeds mainly
on leaf litter mixed with the soil of upper horizons. They
appear to prefer micro-organism-contaminated organic
matter and assimilated micro-organisms (Edwards and
Fletcher, 1988, Zhang et al., in preparation). Our results
are similar to those obtained by UrbaÂsÏek (1990), in the
study of cellulase activity in the earthworm gut, who
found that epigeic species had higher gut cellulase activity
than endogeic earthworms that feed on soil less enriched in
organic matter.
In this study, we did not detect chitinase activity in the gut
of earthworms. In contrast, in the tropical earthworms
Pontoscolex corethrurus and Polypheretima elongata
(Zhang et al., 1993; Lattaud et al., 1997) we found that bN-acetyl-d-glucosaminitase was rather active, chitin is a
component of fungal cell walls and b-N-acetyl-d-glucosamine is the monomer of chitin.
Barois and Lavelle (1986) reported that earthworms
produce a huge amount of intestinal mucus being a mixture
of glyco-proteins, and small glucidic and proteic molecules.
They suggested that the mucus was rapidly incorporated
into microbial biomass in the gut, since no mucus was
recovered in the casts. The weak protease and phosphatase
activity in earthworm casts may have resulted from degradation of the enzymes in the posterior part of the gut. Apart
from degradation of enzymes, low phosphatase activities
may have resulted from inhibition of expression of enzymatic activities by the inorganic phosphate (McGill and
Cole, 1981). However, in our experiment, this was not the
case: the higher content of NaHCO3-extractable inorganic
phosphorus in casts cannot account for the decrease of
phophatase activity (data not shown). Our results contradict
the ®ndings of Sharpley and Syers (1976) who found that
phosphatase activity was higher in fresh casts of earthworms
(L. rubellus and A. caliginosa) than in uningested soils; this
difference may be partly explained by the fact that different
species were used in each experiment.
Acknowledgements
The research was funded by the Natural Science Foundation of China. We thank M. Li Nai-Guang and Li Guo-Xue
for aid in earthworm collection, Dr Patrick Lavelle Professor of the Universite Pierre et Marie Curie and Dr Graham
Sparling, Landcare Research, New Zealand for constructive
critiques and suggestions.
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www.elsevier.com/locate/soilbio
Changes in microbial biomass C, N, and P and enzyme activities in soil
incubated with the earthworms Metaphire guillelmi or Eisenia fetida
Bao-Gui Zhang*, Gui-Tong Li, Tian-Shou Shen, Jian-Kui Wang, Zhao Sun
College of Agricultural Resources and Environment, China Agricultural University, Beijing 100094 People's Republic of China
Accepted 6 June 2000
Abstract
Earthworms are ubiquitous in soil and ingest large amounts of soil, organic matter and leaf litter. To assess changes in organic matter
fractions after passage through the earthworm gut, we measured microbial biomass C, N and P and the fungal-to-bacterial ratios in wormworked soil (WWS), obtained by incubating soil for 24 h with large numbers of the anecic earthworm Metaphire guillelmi (1:5 ratio of fresh
weight worms: dry weight soil). Microbial biomass C, N and P were estimated by the fumigation±extraction methods, and fungal-to-bacterial
ratios by selective inhibition using substrate induced respiration. Enzyme activities in the gut of M. guillelmi were also compared with an
epigeic earthworm species Eisenia fetida. Activities of cellulase, protease, chitinase, acid and alkaline phosphatases, in gut, casts and
uningested soil were measured.
In WWS, microbial biomass decreased (130 mg C g 21 soil), and there was a concomitant increase of available nutrients (27 and 10 mg g 21
soil for ninhydrin-reactive N and inorganic P, respectively). There was no difference between the glucose-sensitive microbial biomass (MB)
and the control but the respiratory quotient was greater (2.85 ^ 0.17 and 2.95 ^ 0.07 mg CO2-C g 21 soil h 21 for WWS and control,
respectively). The fungal-to-bacterial ratio was slightly higher in WWS than in uningested soil (1.61 vs. 1.35).
Cellulase activity was greater in the gut of the epigeic earthworm than in that of the anecic one (152.8 ^ 18.7 vs. 18.9 ^ 1.3 mg
glucose g 21 worm fw h 21); conversely, protease and phosphatase activities were signi®cantly higher in gut of the anecic specie as opposed
to the epigeic species. The activity of cellulolytic enzymes was slightly higher in casts than in soil; while activities of protease, acid (pH 6.5)
and alkaline (pH 9.0) phosphatases were lower in earthworm casts than in the uningested soils (protease, acid and alkaline phosphatase
activity were 29.5 ^ 1.7 mg tyrosine g 21 worm fw h 21, 570 ^ 2.9, 748 ^ 7.3 mg p-nitrophenol g 21 soil h 21 in soil and 17.8 ^ 2.0 mg
tyrosine g 21 worm fw h 21, 327 ^ 26.7, 549 ^ 19.7 mg p-nitrophenol g 21 soil h 21 in casts, respectively). We conclude that micro-organisms
are used by earthworms as a secondary food resource, and that passage through earthworm gut decreases the total soil MB and increase the
active components of MB. q 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Earthworm; Microbial biomass; Protease; Phosphatase; Cellulolytic enzyme; Substrate-induced respiration
1. Introduction
Earthworms are ubiquitous soil animals and dominate the
invertebrate biomass in temperate soils. Worms ingest a
large amount of soil, organic matter and surface litter. In
soils where they are abundant, the surface layers may be
considered as earthworm casts of different age (Lavelle,
1978). Thus earthworms are thought to have a profound
in¯uence on soil organic matter decomposition and nutrient
transformations. Earthworms prefer a diet of decomposed
and decomposing organic matter, which suggests that
micro-organisms are also ingested as food (Edwards and
Fletcher, 1988). As micro-organisms are a key factor in
soil nutrient transformations and the soil microbial biomass
* Corresponding author.
E-mail address: zhangbg@mail.cau.edu.cn (B.-G. Zhang).
(MB) serves both as a pool and as a source of plant nutrients,
ingestion of MB by worms could affect these processes and
nutrient supply. Previous work in this area has given contradictory ®ndings. Plate counts of proteolytic bacteria, total
bacteria and actinomycetes were reported to be increased by
passage through earthworm intestinal tracts (Parle, 1963;
Daniel and Anderson, 1992; Devliegher and Verstraete
1995); while Pedersen and Hendriksen (1993) reported
that numbers of some bacteria increased and others
decreased. MB was reported to decrease (Bohlen and
Edwards, 1995; Devliegher and Verstraete 1995), increase
(Scheu, 1992) or remain unchanged (Daniel and Anderson,
1992) after passage through the earthworm gut.
Little information is available concerning the capacity of
earthworms to degrade organic matter. Most studies on
earthworm digestive enzymes have concentrated on
Lumbricidae (Zhang et al., 1993), with cellulases being
0038-0717/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00111-5
2056
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
the most commonly studied enzymes (Parle, 1963; Loquet
and Vinceslas, 1987; UrbaÂsÏek, 1990). Proteases, phosphatases and chitinases, important in soil N and P transformations, have been less well studied in earthworms.
Our objectives were, ®rstly, to determine the effects of
passage through earthworm guts on the soil microbial
community, especially microbial biomass C, N, P and
respiration, and secondly, to determine the activity of the
digestive enzymes of earthworms and their casts.
2. Materials and methods
2.1. Soil and earthworms
Surface soil (0±10 cm) was taken from the experimental
station in the campus of China Agricultural University,
Beijing. The soil had the following physical and chemical
properties: pH (in H2O) 7.4, clay 16%, silt 41%, sand 43%,
soil organic matter 1.8%, total N 0.1%. The soil was airdried and sieved (,2 mm) and well mixed. Four parts (v/v)
of soil were mixed with one part of sieved (,2 mm) wheat
straw compost; the mixture was adjusted to 50% water holding capacity and were conditioned at 258C at least 10 d
before the experiment began.
Specimens of the anecic earthworms Metaphire guillelmi
(Sims and Easton, 1972) were collected by digging from a
green food production base in a Western suburb of Beijing
and cultivated in the laboratory for 1 month. Adult earthworms, with fresh weight varies between 2.3 and 2.5 g, were
washed free from soil and blotted with ®lter paper before
fresh weight determinations were made. The earthworms
were placed in the incubated mixture for 1 d prior to the
start of the experiment, in order to replace their gut contents
by the mixture. Adult epigeic earthworms Eisenia fetida (fw
varied from 0.4 to 0.5 g) were purchased from the Chinese
Agricultural Academy's laboratory stock.
2.2. Microbial biomass determination
To ensure the soil contained a large proportion of earthworm casts and that the casts in the worm-worked soil
(WWS) could be relatively homogeneous in age, a large
number of earthworms were added to a comparatively
small volume of soil and a short treatment time was chosen.
The gut transit time varies from 2 to 20 h for most species
(Parle, 1963; Lavelle, 1978; Lee, 1985), so a residence time
of 24 h was selected for the experiment. A preliminary
experiment showed that by adding 1 g fresh weight worms
to every 5 g dry weight soil, more than 50% of soil passes
through earthworm intestine during 24 h of treatment.
Although the earthworms were a little crowded, there was
no weight loss during the experiments.
To a sample of 200 g (dw equivalent) of the conditioned
soil, earthworms were added at a ratio of 1 g fresh weight
worms to every 5 g (dw equivalent) soil. After 24 h at 258C,
the earthworms were withdrawn. The worm-worked soil
was further incubated for 3 d (3 d WWS) at 258C with the
soil moisture adjusted to 50% water holding capacity. The
worms removed from the pots were washed free from soil,
weighed and reintroduced into another sample of the conditioned soil. After 24 h, the earthworms were removed, the
WWS were further incubated for 2 d (2 d WWS). The earthworms were washed once more, weighed and re-introduced
into a new sample in order to obtain 1 d WWS. Then, each
WWS sample was homogenised and subsampled in triplicate to determine soil moisture content and measure microbial biomass C, N and P. Worm-free soils were used as the
controls and were treated in the same way as the WWS
samples.
For biomass C and N estimation, triplicate samples of
15 g (dw equivalent) were extracted with 80 ml of 0.5 M
K2SO4 immediately after exposure to worms or after a
subsequent 24 h fumigation with CHCl3. Organic C in the
extracts was measured in 5 ml aliquots by the dichromate
oxidation method (Vance et al., 1987). Ninhydrin-reactive
N (Nnin) in the extracts was analysed by reacting aliquots
(2 ml) with a ninhydrin reagent (Moore and Stein, 1954) and
reading absorbance at 570 nm. Microbial biomass C and N
were calculated, respectively, by the equations Bc EC £
2:64 (Vance et al., 1987) and Bn EN £ 5:0 (Joergensen
and Brookes, 1990), where EC and EN are the difference
between organic C or ninhydrin-reactive N extracted by
0.5 M K2SO4 from fumigated and non-fumigated soil,
respectively.
For microbial biomass P determination, triplicate samples
of 15 g were extracted with 80 ml of 0.5 M NaHCO3 (pH
8.5) immediately at the end of incubation or after a subsequent 24 h fumigation with CHCl3. Inorganic P (Pi) was
analysed in aliquots (1 ml) of the extracts by the ammonium
molybdate±ascorbic acid method described by Murphy and
Riley (1962). A spike of KH2PO4 equivalent to 25 mg P g 21
soil was used to correct for Pi ®xation during the NaHCO3
extraction. Biomass P was calculated by Bp EP =0:38;
where EP is the difference between NaHCO3-Pi extracted
from fumigated and non-fumigated soil (Brookes et al.,
1982). NH41-N and NO32 were measured in the same extracts
as microbial biomass C and N. NH41-N was analysed by
indophenol blue method (Keeney and Nelson, 1982),
NO32-N was analysed by a colorimetric method of nitration
of salicylic acid (Cataldo et al., 1975).
2.3. Substrate-induced respiration (SIR) rate and bacterialto-fungal ratio
Triplicate WWS samples were obtained by exposing
earthworms to conditioned soil for 24 h, at a 1:5 ratio of
worms to soil (fw/dw). Then, SIR was determined in triplicate samples (ca. 10 g dry weight) of WWS or uningested
soil with a continuous air-¯ow system modi®ed and adapted
from Cheng and Coleman (1989). Glucose was added to soil
with talcum as a carrier. Preliminary experiments showed
that 10 mg glucose g 21 soil was optimal for a maximal
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
response. Streptomycin and cycloheximide (Sigma Chemical Co. St Louis, MO) were used as inhibitors of bacterial
and fungal respiration respectively, the inhibitors were used
separately and also combined. A preliminary experiment
showed that 8 mg g 21 was the optimal concentration for
inhibition of respiration, and a greater streptomycin concentration stimulated soil respiration. As streptomycin contains
N, and non-target micro-organisms (e.g. fungi) may use it as
a source of N, in samples not amended with streptomycin, N
was added as (NH4)2SO4 solution allowing all treatments to
have the same quantity of N. The mean respiratory rates of
0.5±2.5 h after addition of glucose and inhibitor(s) were
used to calculate SIR and to evaluate the effects of the
inhibitors. Selectivity of inhibitors was estimated by the
additivity ratio (expressed as sum of reduction in
substrate-induced respiration with separate addition of
streptomycin and cycloheximide divided by reduction
caused by the application of both inhibitors in combination).
The metabolic quotient of the soil MB qCO2 was obtained
by dividing basal respiration rate by total soil MB and
expressed as CO2 evolved per unit microbial C per unit
time (Anderson and Domsch, 1978).
2057
phenol reagent (Lowry et al., 1951) was added and the
mixture was homogenised immediately. Absorbance at
700 nm was determined 20 min later and related to those
of tyrosine standards (0±1000 mM) treated in a similar way.
Acid (pH 6.5) phosphatase activity was determined
according to the method adapted from Tabatabai (1982).
Centrifuged homogenate (500 ml) solution was incubated
with 4 ml of modi®ed universal buffer (MUB) at pH 6.5,
and 1 ml of 25 mM sodium p-nitrophenyl phosphate in pH
6.5 MUB at 378C. After 30 min, the reaction was stopped by
adding 4 ml of 0.5 M NaOH solution. After a further 20 min
at room temperature, the concentration of p-nitrophenol was
determined by reading the absorbance at 420 nm and related
to those of 1% p-nitrophenol standards.
Chitinase activity was determined by incubating 1 ml of
centrifuged homogenate with 1 ml of citrate-phosphate
buffer pH 5.2 and 1 ml of 0.2% chitin suspension for 24 h
(Skujins et al., 1965). After stopping the reaction in boiling
water and centrifugation, liberated b-N-acetyl-d-glucosamine in the supernatant was measured as described by Reissig et al. (1955).
2.5. Enzymatic activity in casts and soil
2.4. Enzymatic activity in gut of the earthworms
Adult earthworms were washed free from soil and incubated in soil or soil/decomposed straw mixture (worm to
soil or mixture at 1:5 ratio, fw/dw) for 24 h. Sub-samples
of fresh casts were collected, the earthworms were washed
and weighed. Four individuals of M. guillelmi were sampled
and dissected. After dissection, the gut wall and its contents
of one M. guillelmi worm were homogenised in a known
volume of 50 mM pH 4.8 citrate buffer at 48C to determine
cellulase activity, or in pH 8.1 Tris±HCl buffer to determine
protease activity. The homogenates were centrifuged for
10 min at 6000 rpm, the supernatant was stored at 2188C
until the enzymes were assayed. Because of their small size,
E. fetida (2 individuals for each replication, 6 replications)
were homogenised without dissection. The enzymatic activities of both species were expressed per unit fresh weight of
worm.
Cellulase activity was determined using a ®lter paper
assay (Mandels et al., 1976). Brie¯y, 1 ml of 50 mM pH
4.8 citrate buffer, 1 ml centrifuged homogenate, 0.5 ml
toluene and a ®lter paper strip were incubated at 508C for
24 h. The reducing-sugar concentration was estimated by
the dinitrosalicylic acid method (Miller, 1969). Glucose
was used as the standard for reducing-sugars.
Protease activity was measured as described by Ladd and
Butler (1972). Centrifuged homogenate (1 ml) was incubated with 2.5 ml of 1% sodium caseinate in 100 mM Tris
buffer pH 8.1 at 508C. After 60 min the reaction was stopped
by precipitating the enzyme and the remaining caseinate by
adding 1 ml of 17.5% trichloroacetic acid. After centrifugation, 2 ml of the supernatant was mixed with 3 ml of 1.4 M
Na2CO3. Then 30 min later, 1 ml of 2-fold diluted Folin-
Protease activity of earthworm casts and soil was determined using the same method as for earthworm extracts.
Preliminary experiments showed that no glucose liberated
from degradation of cellulose or ®lter paper under conditions of our test was detectable by the DNS method (Miller,
1969) or by reducing sugars methods (Smoggyi, 1945).
Thus the CO2 evolution rate from casts and soil incubated
with micro-crystalline cellulose (MCC) was used as an indicator of cellulolytic enzyme activity. Earthworm casts or
soil (20 g) were mixed thoroughly with 200 mg MCC, and
incubated at 258C for 7 d. CO2 evolved was absorbed by
5 ml of 1.0 M NaOH which was replaced daily. Residual
alkali was titrated against 300 mM HCl after precipitating
carbonate by excess of BaCl2, with phenolphthalein as indicator.
Acid (pH 6.5) and alkaline (pH 9.0) phosphatase activities of casts and soil were determined by the method
described by Tabatabai (1982).
Enzymatic activities of earthworms are presented on a
live weight basis, and other results are presented on an
oven-dry soil weight basis. All results are the means of
independent assays (number of mean are indicated in corresponding tables and ®gures). Student's t-test was used to
compare signi®cant difference between the means.
3. Results
3.1. Effect of earthworm on total soil microbial biomass
Soil microbial biomass (MB), C, N and P had decreased
after 24 h of earthworm treatment, while soil extractable
2058
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Table 1
Soil microbial biomass, soluble organic C and available N and P for each treatment. Standard errors of the mean n 3 are shown in parenthesis
Treatment
Control
Earthworm treatment
Microbial biomass a (mg g 21 soil)
Soil extractable C, N and P (mg g 21 soil)
C
N
P
Dichromate-oxidable organic C
Ninhydrin-reactive N
NaHCO3-inorganic P
920.6 (67.5)
791.1 (11.1)
*b
205.25 (3.4)
146.35 (10.9)
***
102.9 (6.6)
74.9 (1.7)
**
84.6 (7.6)
110.7 (7.9)
*
10.43 (0.02)
37.54 (0.29)
***
94.3 (1.6)
104.1 (1.6)
**
a
Kc for biomass C, N, P were used as 0.41, 0.2 and 0.37, respectively.
*, ** and *** represent, respectively, P , 0:05; P , 0:01 and P , 0:001 probability of signi®cant difference between earthworm treatment and control (ttest).
b
organic C, ninhydrin-reactive N (Nnin) and NaHCO3-Pi had
increased (Table 1).
In the WWS, Nmin consisted mainly of NH41-N, which was
nitri®ed during the subsequent incubation as indicated by an
increase of soil NO32-N (Fig. 1). Inorganic P was higher in
WWS than in the control soil and the amounts increased
during the subsequent incubation (Fig. 2).
combined inhibition reached 41% for the earthworm treatment and 48% for the control. Inhibitor selectivity was satisfactory, as the inhibitor additivity ratio was close to 1.0
(1.12 and 1.04 for control and WWS, respectively).
3.2. Effect of earthworms on the soil microbial community
Cellulase activity was 7-fold greater in the guts of E.
fetida than that in the guts of M. guillelmi (Table 2). In
contrast, protease and acid phosphatase activity of E. fetida
were signi®cantly lower than in M. guillelmi
(135.5 ^ 1.2 mg tyrosine g 21 fw h 21, 474 ^ 0.11 mg pnitrophenol g 21 fw h 21) (Table 2).
Chitinase activity was not detected in the gut of either of
the two earthworm species.
Although the total MB decreased during the earthworm
treatment, the glucose-sensitive component of the MB was
not considerably affected, as there was no signi®cant difference in glucose-induced respiration between the earthworm
treatment and the control (2.85 ^ 0.17 and 2.95 ^ 0.07 mg
CO2-C g 21 soil h 21 for WWS and control, respectively).
The Metabolic quotient (qCO2), was signi®cantly P ,
0:05 higher in WWS than in the control soil (10.66 ^ 1.9
and 5.09 ^ 0.48 mg CO2-C mg 21 microbial biomass-C h 21
for WWS and control, respectively). This resulted from a
combination of an increase of basal respiration and a decline
of MB in the earthworm treatment. The fungal-to-bacterial
ratio of the glucose-sensitive MB was slightly higher in
WWS than in the uningested soil (1.61 vs. 1.35). The total
Fig. 1. Effects of earthworm on soil ninhydrin N and mineral N. Bars
represent standard errors.
3.3. Cellulase, protease and phosphatase activities in
earthworm gut
3.4. Digestive enzymes in soil and casts of Metaphire
guillelmi
Activities of protease, acid and alkaline phosphatases in
the casts of the earthworm M. guillelmi were 17.8 ^ 2.0 mg
tyrosine g 21 soil h 21, 327 ^ 26.7 and 549 ^ 19.7 mg pnitrophenol g 21 soil h 21, respectively, while those in the
Fig. 2. Effect of earthworm on soil inorganic P. Bars represent standard
errors.
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
2059
Table 2
Activity of digestive enzymes in gut of two earthworm species, means and standard errors and numbers of the mean (in parenthesis)
Enzymatic activity a
Earthworm
E. fetida
M. guillelmi
a
b
Cellulase
Protease
Phosphatase
152.8 18:7; n 6
18.9 1:3; n 4
*** b
24.6 0:3; n 4
135.5 1:2; n 4
****
127.0 0:11; n 3
473.9 4:9; n 3
**
Expressed respectively as mg glucose, mg tyrosine, and mg p-nitrophenol produced g 21 earthworm live weight h 21.
**, ***, ****: signi®cantly different between E. fetida and M. guillelmi at P , 0:001; P , 0:0001 and P , 0:00001; respectively (t-test).
control soil were 29.5 ^ 1.7 mg tyrosine g 21 soil h 21,
570 ^ 3 and 748 ^ 7.3 mg p-nitrophenol g 21 soil h 21,
respectively (Table 3).
The CO2 evolution rates were higher in casts than in the
control soil when a mixture of soil and decomposed wheat
straw was used as the medium for earthworm culture, which
suggested that the microbial activity was greater in casts
compared to the control soil (Fig. 3). The difference of the
CO2 evolution rate between worm casts and soil was maximum at day 5, with the CO2 evolution rate of the casts being
3-fold greater than that in soil. Cellulose amendment did not
further increase CO2 evolution rate either in earthworm
casts or in the control soils.
When the earthworm culture medium was soil (without
wheat straw addition), in the absence of cellulose, there was
no difference of CO2 evolution rate between casts and uningested soils, except on day 2, where there was a slight
increase in casts; however, cellulose amendment caused a
higher CO2 evolution rate in earthworm casts compared to
the uningested soils (Fig. 4).
4. Discussion
4.1. Effects of earthworm on micro-organisms
Our data and published information shows that various
components of soil MB respond differently to passage
through earthworm gut. This partly explains why different
results were obtained depending upon which components of
the biomass were measured. Total soil MB is assessed by
Table 3
Activity of digestive enzymes in soil and in fresh casts of Metaphire guillelmi Means n 4 and standard errors of the mean (in parenthesis)
Phosphatase activities (mg pnitrophenol g 21 soil h 21)
Treatments
Soil
Casts
a
Acid
570 (2.9)**
327 (27)
Protease
activity (mg
tyrosine g 21
soil h 21)
Alkaline
a
748 (7.3)***
549 (19.7)
29.5 (1.7)**
17.8 (2.0)
**, ***: signi®cantly different between soil and casts at P , 0:01 and
P , 0:001; respectively (t-test).
chloroform-fumigation methods, SIR method measures the
active MB or more exactly the substrate sensitive component of MB, while plate counting methods provide a
measure of the number of cultivable (or viable) microorganisms. In our study, passage through the earthworm
gut reduced total soil MB (Table 1), which is in accordance
with results reported by Devliegher and Verstraete (1995)
and Bohlen and Edwards (1995). These authors also used
fumigation technique to determine MB. However, Daniel
and Anderson (1992) found no change of soil MB after
gut passage although they used the same fumigation-extraction method. In their experiment, the comparison of MB was
made between earthworm casts and uningested soil; but that
may be an effect of the selective behaviour of earthworms
on soil MB. Earthworms selectively consume soil fractions
with high concentration of micro-organisms (Hendriksen,
1990), and not all the ingested micro-organisms are killed
during passage through earthworm intestine (Edwards and
Fletcher, 1988; Hendriksen, 1990). In that study, the detrimental effect of the earthworm gut on soil MB would have
been counteracted by enriching casts with micro-organisms
through selective feeding behaviour (Hendriksen, 1990). In
our study, soil MB was compared between WWS and the
uningested soils, thus the indirect effect of earthworm selective feeding was eliminated.
In WWS, a reduction in soil MB with a concomitant
increase of extractable organic C, mineral N and
NaHCO3-extractable Pi indicated that release of microbially
immobilised nutrients was enhanced by earthworms (Table
1). The increase in metabolic quotient qCO2 of soil MB in
the WWS suggested a rejuvenation of the microbial community, as the qCO2 quotients of ªyoungº micro-organisms are
frequently greater than that of ªagedº ones (Anderson and
Domsch, 1978). The release of immobilised nutrients may
have a stimulating effect on microbial activity and activate
dormant microbes. Fischer et al. (1997) demonstrated that
passage of microbes through earthworm guts resulted in a
signi®cant increase in the number of vegetative cells and a
decrease in the number of spores of Bacillus megaterium.
Their results suggested that germination of bacterial spores
were enhanced by the gut passage.
In summary, soil total MB was negatively affected by
passage through earthworms gut while active MB (glucose
sensitive MB) increased. Release of microbially-immobilised
2060
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Fig. 3. CO2 evolution rate of soil and casts. Soil was amended with a wheat
straw residue (soil/residue ratio 4:1, w/w) and incubated for 24 h with or
without earthworms (soil:worm ratio 5 dw/fw) without (A) or with (B)
cellulose amendment (avicel, 1% w/w).
nutrients, rejuvenation of the microbial community and
possibly activation of dormant microbes may have
enhanced the microbial activity in the earthworm casts.
Reduction of soil MB by the earthworm M. guillelmi
indicates that this earthworm may consume soil microorganisms. Compared to the epigeic earthworm E. fetida
which feeds on plant litter, the gut of M. guillelmi has
much greater protease and phosphatases activities and
much lower cellulolytic enzyme activities (Table 3). This
suggests that M. guillelmi may assimilate proteins and
organic P compounds in the cells of soil micro-organisms
rather than the cellulose of plant litter. The preference for
partially decomposed organic materials rich in micro-organisms rather than for fresh organic residues by earthworms
(Cortez et al., 1989; Moody et al., 1995) is also an indication
that earthworms may consume micro-organisms. In the
course of organic residue decomposition, exhaustion of
more easily decomposable constituents such as sugar and
proteins precedes that of cellulose and hemicellulose.
Substances remaining in partially decomposed plant residues are mainly cellulose, hemicellulose, lignin, substances
newly synthesised by micro-organisms and microbial cells.
Lignins are very resistant to microbial attack, and microbial
metabolites are thought to be more recalcitrant than the
initial substances. Since cellulase activities were very
weak in the guts of M. guillelmi, microbial biomass must
be the only substrate for the earthworms. Thus it is easy to
speculate that plant residues are used as primary resources
by micro-organisms which in turn, are used by M. guillelmi
as a secondary resource. As for E. fetida, the relatively
higher cellulase activity in the gut may allow them to utilise
plant materials directly as a food source. The results of the
enzymatic activities in the gut and casts of M. guillelmi
suggested that proteins and phosphorus-containing organic
compounds are digested in the gut rather than in the casts,
whilst cellulose seems to mainly be degraded in casts.
Consumption of micro-organisms by earthworms may
have various consequences on organic matter decomposition. Release of microbial immobilised nutrients and rejuvenation of soil MB may enhance the decomposition of soil
organic matter as metabolic activities of micro-organisms
remain intense. When earthworm feeding intensity is high
and soil nutrients are limited, earthworm feeding may have
detrimental effects on microbial population and activity,
consequently the decomposition rate of organic residues
may be reduced. Although the soil fungal-to-bacterial
ratio was not signi®cantly modi®ed by earthworms in our
experiment, there is other evidence (Pedersen and Hendriksen, 1993; Toyata and Kimura, 1994) that the composition
and succession of the microbial community may be changed
by earthworms. Earthworm preferentially feed on fungi
degrading water-soluble sugars and cellulose, while the
lignin-degrading fungi characteristic of the later stage of
plant litter decomposition were less selected (Moody et
al., 1995). It is well known that the capacity to decompose
complex organic matter varies with microbial community
(Campbell, 1983).
4.2. Digestive enzymes in gut and casts of the earthworms
Fig. 4. CO2 evolution rate of soil. Soil was incubated for 24 h with (- - -) or
without (±) earthworms M. guillelmi (soil:worm ratio 5 dw/fw), with (1)
or without (2) cellulose amendment (avicel, 1% w/w).
The difference in the enzymatic activities of the two
earthworms studied may be associated with their ecological
categories and feeding habitats. The earthworm E. fetida is
an epigeic species which feeds mainly on plant litter. Their
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
high cellulase activity allows them to ef®ciently assimilate
plant materials, which are composed mainly of cellulose;
while M. guillelmi is an anecic species which feeds mainly
on leaf litter mixed with the soil of upper horizons. They
appear to prefer micro-organism-contaminated organic
matter and assimilated micro-organisms (Edwards and
Fletcher, 1988, Zhang et al., in preparation). Our results
are similar to those obtained by UrbaÂsÏek (1990), in the
study of cellulase activity in the earthworm gut, who
found that epigeic species had higher gut cellulase activity
than endogeic earthworms that feed on soil less enriched in
organic matter.
In this study, we did not detect chitinase activity in the gut
of earthworms. In contrast, in the tropical earthworms
Pontoscolex corethrurus and Polypheretima elongata
(Zhang et al., 1993; Lattaud et al., 1997) we found that bN-acetyl-d-glucosaminitase was rather active, chitin is a
component of fungal cell walls and b-N-acetyl-d-glucosamine is the monomer of chitin.
Barois and Lavelle (1986) reported that earthworms
produce a huge amount of intestinal mucus being a mixture
of glyco-proteins, and small glucidic and proteic molecules.
They suggested that the mucus was rapidly incorporated
into microbial biomass in the gut, since no mucus was
recovered in the casts. The weak protease and phosphatase
activity in earthworm casts may have resulted from degradation of the enzymes in the posterior part of the gut. Apart
from degradation of enzymes, low phosphatase activities
may have resulted from inhibition of expression of enzymatic activities by the inorganic phosphate (McGill and
Cole, 1981). However, in our experiment, this was not the
case: the higher content of NaHCO3-extractable inorganic
phosphorus in casts cannot account for the decrease of
phophatase activity (data not shown). Our results contradict
the ®ndings of Sharpley and Syers (1976) who found that
phosphatase activity was higher in fresh casts of earthworms
(L. rubellus and A. caliginosa) than in uningested soils; this
difference may be partly explained by the fact that different
species were used in each experiment.
Acknowledgements
The research was funded by the Natural Science Foundation of China. We thank M. Li Nai-Guang and Li Guo-Xue
for aid in earthworm collection, Dr Patrick Lavelle Professor of the Universite Pierre et Marie Curie and Dr Graham
Sparling, Landcare Research, New Zealand for constructive
critiques and suggestions.
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