Changes in microbial biomass C, N, and P and enzyme activities in soil
incubated with the earthworms
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
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 worm-worked soil (WWS), obtained by incubating soil for 24 h with large numbers of the anecic earthwormMetaphire 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 ofM. guillelmiwere also compared with an epigeic earthworm speciesEisenia fetida. Activities of cellulase, protease, chitinase, acid and alkaline phosphatases, in gut, casts and uningested soil were measured.
In WWS, microbial biomass decreased (130mg C g21soil), and there was a concomitant increase of available nutrients (27 and 10mg g21 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 2.95^0.07mg CO2-C g21 soil h21 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. 18.9^1.3mg glucose g21worm fw h21); 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 g21 worm fw h21, 570
^2.9, 748^7.3mg p-nitrophenol g21 soil h21 in soil and 17.8
^2.0 mg tyrosine g21worm fw h21, 327^26.7, 549^19.7mgp-nitrophenol g21soil h21in 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.q2000 Elsevier Science Ltd. All rights reserved.
Keywords: Earthworm; Microbial biomass; Protease; Phosphatase; Cellulolytic enzyme; Substrate-induced respiration
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
(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 contra-dictory ®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 matterq2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 0 ) 0 0 1 1 1 - 5
* Corresponding author.
the most commonly studied enzymes (Parle, 1963; Loquet and Vinceslas, 1987; UrbaÂsÏek, 1990). Proteases, phospha-tases and chitinases, important in soil N and P transforma-tions, 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
air-dried 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
hold-ing capacity and were conditioned at 258C at least 10 d
before the experiment began.
Specimens of the anecic earthwormsMetaphire 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 earth-worms, 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 earthwormsEisenia 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 earth-worm casts and that the casts in the earth-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 condi-tioned soil. After 24 h, the earthworms were removed, the WWS were further incubated for 2 d (2 d WWS). The earth-worms 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 tripli-cate to determine soil moisture content and measure micro-bial 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 BcEC£
2:64 (Vance et al., 1987) and BnEN£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,
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
subse-quent 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 KH2PO4equivalent to 25mg P g21
soil was used to correct for Pi®xation during the NaHCO3
extraction. Biomass P was calculated by BpEP=0:38;
where EP is the difference between NaHCO3-Pi extracted
from fumigated and non-fumigated soil (Brookes et al.,
1982). NH41-N and NO32were 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 bacterial-to-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 tripli-cate 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 g21 soil was optimal for a maximal
response. Streptomycin and cycloheximide (Sigma Chemi-cal 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 g21 was the optimal concentration for
inhibition of respiration, and a greater streptomycin concen-tration 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)2SO4solution 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 MBqCO2was 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).
2.4. Enzymatic activity in gut of the earthworms
Adult earthworms were washed free from soil and incu-bated 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 ofM. guillelmiwere sampled
and dissected. After dissection, the gut wall and its contents
of oneM. 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 at2188C
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 activ-ities 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 incu-bated 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 centrifuga-tion, 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-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±1000mM) treated in a similar way.
Acid (pH 6.5) phosphatase activity was determined according to the method adapted from Tabatabai (1982).
Centrifuged homogenate (500ml) solution was incubated
with 4 ml of modi®ed universal buffer (MUB) at pH 6.5,
and 1 ml of 25 mM sodiump-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 ofp-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-glucosa-mine in the supernatant was measured as described by Reis-sig et al. (1955).
2.5. Enzymatic activity in casts and soil
Protease activity of earthworm casts and soil was deter-mined using the same method as for earthworm extracts. Preliminary experiments showed that no glucose liberated from degradation of cellulose or ®lter paper under condi-tions of our test was detectable by the DNS method (Miller, 1969) or by reducing sugars methods (Smoggyi, 1945).
Thus the CO2evolution rate from casts and soil incubated
with micro-crystalline cellulose (MCC) was used as an indi-cator 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
Acid (pH 6.5) and alkaline (pH 9.0) phosphatase activ-ities 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
corre-sponding tables and ®gures). Student's t-test was used to
compare signi®cant difference between the means.
3.1. Effect of earthworm on total soil microbial biomass
organic C, ninhydrin-reactive N (Nnin) and NaHCO3-Pihad
increased (Table 1).
In the WWS, Nminconsisted 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).
3.2. Effect of earthworms on the soil microbial community
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 differ-ence in glucose-induced respiration between the earthworm
treatment and the control (2.85^0.17 and 2.95^0.07mg
CO2-C g21 soil h21 for WWS and control, respectively).
The Metabolic quotient (qCO2), was signi®cantly P,
0:05higher in WWS than in the control soil (10.66^1.9
and 5.09^0.48mg CO2-C mg21microbial biomass-C h21
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
combined inhibition reached 41% for the earthworm treat-ment and 48% for the control. Inhibitor selectivity was satis-factory, as the inhibitor additivity ratio was close to 1.0 (1.12 and 1.04 for control and WWS, respectively).
3.3. Cellulase, protease and phosphatase activities in earthworm gut
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 ofE. fetida
were signi®cantly lower than in M. guillelmi
(135.5^1.2 mg tyrosine g21 fw h21, 474^0.11mg p
-nitrophenol g21fw h21) (Table 2).
Chitinase activity was not detected in the gut of either of the two earthworm species.
3.4. Digestive enzymes in soil and casts of Metaphire guillelmi
Activities of protease, acid and alkaline phosphatases in
the casts of the earthwormM. guillelmiwere 17.8^2.0 mg
tyrosine g21 soil h21, 327^26.7 and 549^19.7 mg p
-nitrophenol g21 soil h21, respectively, while those in the
B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Soil microbial biomass, soluble organic C and available N and P for each treatment. Standard errors of the mean n3are shown in parenthesis
Treatment Microbial biomassa(mg g21soil) Soil extractable C, N and P (mg g21soil)
C N P Dichromate-oxidable organic C Ninhydrin-reactive N NaHCO3-inorganic P
Control 920.6 (67.5) 205.25 (3.4) 102.9 (6.6) 84.6 (7.6) 10.43 (0.02) 94.3 (1.6) Earthworm treatment 791.1 (11.1) 146.35 (10.9) 74.9 (1.7) 110.7 (7.9) 37.54 (0.29) 104.1 (1.6)
*b *** ** * *** **
a Kc for biomass C, N, P were used as 0.41, 0.2 and 0.37, respectively.
b*, ** and *** represent, respectively,P,0:05;P,0:01 andP,0:001 probability of signi®cant difference between earthworm treatment and control (t -test).
Fig. 1. Effects of earthworm on soil ninhydrin N and mineral N. Bars represent standard errors.
control soil were 29.5^1.7 mg tyrosine g21 soil h21,
570^3 and 748^7.3 mg p-nitrophenol g21 soil h21,
respectively (Table 3).
The CO2evolution 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
CO2evolution rate between worm casts and soil was
maxi-mum at day 5, with the CO2evolution 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 CO2evolution rate between casts and
unin-gested soils, except on day 2, where there was a slight increase in casts; however, cellulose amendment caused a
higher CO2evolution rate in earthworm casts compared to
the uningested soils (Fig. 4).
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
chloroform-fumigation methods, SIR method measures the active MB or more exactly the substrate sensitive compo-nent of MB, while plate counting methods provide a measure of the number of cultivable (or viable) micro-organisms. 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-extrac-tion 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 detri-mental 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 selec-tive feeding was eliminated.
In WWS, a reduction in soil MB with a concomitant increase of extractable organic C, mineral N and
NaHCO3-extractable Piindicated that release of microbially
immobilised nutrients was enhanced by earthworms (Table
1). The increase in metabolic quotientqCO2of soil MB in
the WWS suggested a rejuvenation of the microbial
commu-nity, as theqCO2quotients 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 B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Activity of digestive enzymes in gut of two earthworm species, means and standard errors and numbers of the mean (in parenthesis)
Earthworm Enzymatic activitya
Cellulase Protease Phosphatase
E. fetida 152.8 18:7;n6 24.6 0:3;n4 127.0 0:11;n3
M. guillelmi 18.9 1:3;n4 135.5 1:2;n4 473.9 4:9;n3
***b **** **
a Expressed respectively asmg glucose, mg tyrosine, andmgp-nitrophenol produced g21earthworm live weight h21.
b **, ***, ****: signi®cantly different betweenE. fetidaandM. guillelmiatP,0:001;P,0:0001 andP,0:00001;respectively (t-test).
Activity of digestive enzymes in soil and in fresh casts ofMetaphire guil-lelmiMeans n4and standard errors of the mean (in parenthesis)
Phosphatase activities (mgp -nitrophenol g21soil h21)
Protease Casts 327 (27) 549 (19.7) 17.8 (2.0)
a **, ***: signi®cantly different between soil and casts atP
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
micro-organisms. 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-organ-isms 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 resi-dues 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 byM. 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 decomposi-tion. Release of microbial immobilised nutrients and reju-venation 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 Hendrik-sen, 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
The difference in the enzymatic activities of the two earthworms studied may be associated with their ecological
categories and feeding habitats. The earthwormE. fetidais
an epigeic species which feeds mainly on plant litter. Their B.-G. Zhang et al. / Soil Biology & Biochemistry 32 (2000) 2055±2062
Fig. 3. CO2evolution rate of soil and casts. Soil was amended with a wheat straw residue (soil/residue ratio4:1, w/w) and incubated for 24 h with or without earthworms (soil:worm ratio5 dw/fw) without (A) or with (B) cellulose amendment (avicel, 1% w/w).
high cellulase activity allows them to ef®ciently assimilate plant materials, which are composed mainly of cellulose;
whileM. guillelmiis 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 thatb
-N-acetyl-d-glucosaminitase was rather active, chitin is a
component of fungal cell walls andb-N
-acetyl-d-glucosa-mine 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 degra-dation 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 enzy-matic 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. rubellusandA. caliginosa) than in uningested soils; this difference may be partly explained by the fact that different species were used in each experiment.
The research was funded by the Natural Science Founda-tion of China. We thank M. Li Nai-Guang and Li Guo-Xue for aid in earthworm collection, Dr Patrick Lavelle Profes-sor of the UniversiteÂ Pierre et Marie Curie and Dr Graham Sparling, Landcare Research, New Zealand for constructive critiques and suggestions.
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