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Microbial biomass and activity in contrasting soil materials after passage through the gut of the earthworm Lumbricus rubellus hoffmeister
Soil Biol. Biochem. Vol. 24, No. 5, pp. 465470, Printed in Great Britain. All rights reserved
1992 Copyright
0
0038-0717/92 $5.00 + 0.00 1992 Pergamon Press Ltd
MICROBIAL BIOMASS AND ACTIVITY IN CONTRASTING SOIL MATERIALS AFTER PASSAGE THROUGH THE GUT OF THE EARTHWORM LUMBRICUS RUBELLUS HOFFMEISTER 0. DANIEL’* and J. M. ANDERSON’ I Institute of Terrestrial Ecology/Soil Biology, ETH, CH-8952 Schlieren, Switzerland and *Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter EX4 4PS, U.K. (Accepted
25 November
1991)
Summary-Earthworms (Lumbricus rubehs) were fed on four different soils with light fraction organic material contents from 3.7 to 76.1% of the soil dry weight, and soil water potentials standardized at - 8 kPa. Microbial biomass-C in the soils, as measured with a modified fumigation
INTRODUCTION
MATERIALS AND
METHODS
Soils
In temperate and tropical ecosystems earthworms may ingest large amounts of mineral soil, soil organic matter and surface litter, and produce casts deposited below-ground and on the surface up to as much as 270 t ha-’ yr-t (Lee, 1985). Soil profile development may be influenced by earthworms (Nielson and Hole, 1964), and surface layers of some soils in Europe
(Kubiena, 1953) and African savannas (Lavelle, 1978) may consist almost entirely of earthworm casts. Comparisons between soils and casts indicated that passage through the earthworm gut usually results in higher plate counts of bacteria (Satchell, 1983) and increased rates of microbial respiration (Parle, 1963; Scheu, 1987a). Rates of nitrification (Parle, 1963; Syers et al., 1979; Scheu, 1987b) and denitrification (Elliott et al., 1990, 1991) may also be higher in casts than in bulk soil, indicating that microbial activity is generally stimulated by gut passage. Since microbial biomass is widely used as a key factor mediating soil processes (Jenkinson and Ladd, 1981), we have investigated whether the high microbial activity reported in earthworm casts were related to an increase in the microbial biomass. In order to carry out the measurements the method of Vance et al. (1987) for determining the microbial biomass was callibrated for small soil and cast samples. Four soils with different organic matter content were then used to test the influence of the passage through the gut of Lumbricus rubellus Hoffmeister on the microbial biomass, and related measurements of microbial respiration, bacterial plate counts, soluble organic-C and moisture content.
*Author for correspondence. 465
The soils used were taken from beneath the root mat in a lawn, from a tilled garden plot, and from the humus layer of beech and spruce forests. Aggregates were gently broken down by hand, and stones and roots were removed. The materials were sieved (< 5 mm) and stored in containers at 2-3°C. The soils were prepared for feeding experiments by standardizing moisture contents at - 8 kPa. This involved firming the soil into 4.8 cm dia porous ceramic cups (Soilmoisture Equipment Corp.) to a depth of ca 3 cm, ponding three times with distilled water and allowing equilibrium to be reached with the applied suction tension (80cm water column) over 24 h. The soil moisture content was determined by gravimetry, after drying for 24 h at 105°C. Thereafter, soils stored in containers were carefully moistened with a volume of distilled water sufficient to restore the moisture content to a level equivalent to a water potential of - 8 kPa. The light-fraction (LF) material was separated from the soils by the method of Strickland and Sollins (1987), which was modified for small soil samples. Aliquots of 14 g dry soil (24 h at 105C) were placed in 25 ml universal bottles and dispersed by shaking in I5 ml NaI solution (density = 1.7 g cm-‘) for 1 h on an orbital shaker. The heavy-fraction was allowed to settle for 24 h at room temperature. The LF material on the surface of the NaI solution was transferred by spatula and by aspiration onto a tared, oven-dry glass fibre filter (Whatman GF/C), washed three times by filtration with lOm1 1 M NaCl and three times with distilled water. The weight was then determined after drying 24 h at 105°C. The soil pH was measured in the supernatant of a 1: 2.5 (v: v) soil: 10 mM CaCl, suspension, after shaking and allowing the soil to settle for 3 h.
466
calibration
0. DANIEL and J.M.
of‘ the fumigation-e.~tract~on
inet~lod
The method of Vance et al. (1987) has been used extensively to measure the microbial biomass in a r-angc of soil types (Sparling and West, 1988; Tate (lt ul., 1988; Martikainen and Palojlrvi, 1990; Ocio and Brookes, 1990; Vance and Nadkarni, 1990) and an automated procedure (Wu er al., 1990) has been developed to reduce the time taken for measurements. In our study this method had to be modified for small soil samples such as earthworm casts. Moist soils (equivalent to SO-250 mg dry wt) were weighed into IO ml universal bottles. Non-fumigated soils were extracted immediately, as described below. For the fumigation. the universal bottles were placed in a desiccator lined with wet filter paper to maintain humidity, and containing about 25 ml distilled CHCl, in a flask with anti-bumping granules. The desiccator was evacuated by a vacuum pump until the CHCI, boiled for about 2 min. The evacuated desiccator was then placed in the dark at 25’C. After 24 h the flask with the CHCI, and the filter paper was removed and the desiccator was evacuated 6 times for S min and flushed with air to remove the remaining CHCl, vapour. For the extraction, 4ml 0.5 M K,SO, was added. and the universal bottles were shaken for I h on an orbital shaker. The soil suspension was then filtered through glass fibre filters (Whatman, GF/C) directly into Pyrex 100 ml block digester tubes, with a further two aliquots of 2 ml 0.5 M KzSO, to rinse the universal bottles. Since varying amounts of the extraction solution (depending on soil and quantity) remained in the filters, the volume of the extract in the digester tubes was calculated based on its weight and the density of 0.5 M K?SO, (1.145 g cm-‘). It was assumed that the solution had the same organic-C concentration in the filters and in the digester tubes. The filtered extracts were digested with 10 ml H,SO, (98%), 5 ml H,PO, (SS%), and 2mi 33.3 rnM (fumigated soils) or 16.7 mM (non-fumigated soils) NazCrzO, in a block digester (Kjeldatherm”, Gerhardt GmbH, Bonn) that had been heated to 17O’C. After 30 min the tubes were removed from the block digester, allowed to cool and their contents were quantitatively transferred into 100 ml Erlenmeyer flasks, using distilled H,O to bring the volume to about SO ml. The excess dichromate was determined by backtitration with 16 and 8rn~ ferrous ammonium sulphate in 0.4 M H,SO, for fumigated and respectively. N-phenylannon-fumigated soils, thranilic acid was used as indicator. Losses of dichromate, which are proportional to the amount not used for the oxidation of the organicC in the sample, occur by boiling in a bfock digester; this proportion can be estimated with boiled and unboiled controls (Yeomans and Bremner, 1988). at the low organic-C concentrations However, measured here, constant dichromate losses, due to organic-C from the filters or the chemicals used, may also occur. Since proportional and constant effects
ANDERSON
cannot be separated, a calibration curve with glucose was determined for each digestion run, using 8 concentrations which theoretically used between O-60% of the added dichromate. Microbial biomass-C was calculated as the difference between the organic-C extractable after fumigation and the organic-C extractable without fumigation (soluble-C) multiplied by 2.64 (Vance et al., 1987). This procedure was first validated with the four different soils, determining the extractable-C both with and without fumigation in 5-6 soil portions with dry weights between 50 and 250mg before use on experimental materiais. CO, production CO2 production from casts and soils was measured within 25 ml universal bottles, sealed with SubaSeal bungs, and containing 5 ml 4 mM KOH to absorb the CO: and the sample in a 4mm dia specimen tube attached to the SubaSeal bung. Samples of 20-200 mg dry weight cast or soil were incubated for 48 h in the dark at 25C. The absorbed CO* was then precipitated by 0.4 ml 1 M BaC&, 4 drops of phenolphthalein were added as indicator and the remaining KOH was titrated with 2mM HCl using a microburette. Solutions at working strength were used immediately and at least two blanks were measured per IO samples. Bacterial dilution plate counts Soil and cast samples of 20-200 mg dry wt were dispersed in 10 ml sterile l/4 strength Ringers solution by ultrasonicating at 45 kHz for 4 min, and serial tenfold diluted. One replicate per dilution (0.1 ml) was spread on Oxoid nutrient broth agar plates and incubated for 36-48 h at 2O’C. Zncubation of soils and earth~lor~s To ensure that the animals were in a similar physiological condition for the experiments, L. rubellus was kept for several weeks before the use at 15C in the dark in lawn soil. For the experiments single adult, or subadult, L. rubellus were added to 9 cm dia Petri dishes containing 15-20 g of soil (- 8 kPa water potential) which had been gently compressed to consolidate the structure. Twenty Petri dishes for each of the four soils were placed in covered boxes lined with wet filter paper and kept in the dark at 15°C. After 6-8 h, when the colour of the casts changed except for in lawn soil, all casts were removed from the soil surface. The Petri dishes with the earthworms were incubated a further 16-20 h (i.e. casts had a mean age of 9 h). Soil and casts were sampled then from five Petri dishes each, to compare the microbial biomass-C, organic soluble-C, CO& production, % moisture content and bacteria1 plate counts in casts and bulk soil. Data were transformed with X’ = log(X -t l), where X means microbial biomass-C, organic soluble-C, CO,-C production, % moisture content or bacterial plate counts and X’ is
Microbial
biomass
and activity
467
in soils: influence of earthworms
Table I. Regressions of organic-C (mg gg’) extracted from a range of 6 soil samples (S&250 mg dry wt) before and after chloroform fumigation. Soils were collected from beneath a grass ward (lawn), a tilled garden plot and from beech and spruce forests Extractable-C without fumigation
Lawn Garden Beech forest Spruce forest
usP >
Intercept
Slope
0.003N” O.OlONs 0.042NS 0.044*
0.172*** 0.543*** 0. I 82Ns 0.601***
0.05; ‘P < 0.05; ***p
4
I= 0.801 0.956 0.342 0.860
Extractable-C after fumigation -. 0.067NS 0.017NS -0.042NS - 0.049NS
r2
SlOpe
Intercept
0.570”’ 0.985*** 2.714’** 5.150***
0.568 0.948 0.931 0.992
0.001.
the transformed value, to improve the homogeneity of variances, and the significance of the influence of soil origin and a passage through the gut of L. rubellus was tested by two-factor analyses of variance (Zar, 1984). RESULTS
The modified fumigation-extraction method allowed the determination of extractable organic-C in 50-250mg dry wt soil both before and after CHCI, fumigation. Intercepts of regression lines were below 5Opg C g-’ and, except for the non-fumigated soil from the spruce forest, not significantly different from 0 (Table 1). Slopes of regression lines were significantly different from 0, except for the non-fumigated soil from the beech forest. Coefficients of determination were low for regressions for non-fumigated soil from the beech forest (0.342) and for fumigated soil from the lawn (0.568), but above 0.8 for the other regressions. The LF of the soils increased in the order lawn-garden-beech--spruce over a range from 3.7 to 76.1% of the soil dry weight (Table 2). Assuming that organic matter has 52.6% organic-C (Nelson and Sommers, 1982), the LF organic-C content in the four soils were calculated as respectively 19.5, 23.2, 145.8 and 400.5 mg C g-’ soil. The forest soils with a high LF content were acid, soils from lawn and garden were both in the neutral pH range. Moisture content of the soils at -8 kPa ranged from 38.7 to 132.9%. Microbial biomass-C [Fig. l(a)] and moisture content [Fig. l(b)] in the bulk soil increased with increasing LF content. Microbial biomass-C as % of LF organic-C was 2.1, 8.8, 3.1 and 1.9% in the soils from lawn, garden, and beech and spruce forest, respectively. Soil respiration rate [Fig. l(c)] also increased with increasing LF content, except for the spruce forest soil, where it was lower than in the soil from the beech forest. Amounts of soluble organic-C [Fig.
l(d)] and bacterial plate counts [Fig. l(e)] indicated no relation to the LF content. The origin of the soils had a greater effect on the measured soil properties than a gut passage. This is shown by the relative importance of treatment effects (expressed as treatment SS as % of total SS) which ranged from 41.1 to 89.3% for soil origin and from 1.4 to 24.4% for the gut passage (Table 3). The effect of gut passage on microbial biomass-C was the only treatment which was not significant. DISCUSSIOFi
Microbial biomass measurements by substrate induced respiration (Anderson and Domsch, 1978) fumigation-incubation (Jenkinson and Powlson, 1976) and fumigationextraction (Vance et al., 1987) are usually made on soil samples of 1OOg (wet wt), 250 g (wet wt) and 50 g (dry wt), respectively. Our demonstration that microbiat biomass can be determined on small soil samples of 50-2.50 mg (dry wt) offers the possibility of investigating many processes mediated by invertebrate-microbial interactions operating at this microsite level (Anderson, 1988). Regressions of extractable-C with intercepts being not different from 0 and slopes being different from 0, with one exception each, indicate that the method used is basically sound. For the calibration of nonfumigated soil from the beech forest and for fumigated soil from the lawn, slightly higher soil weights might have been more appropriate to locate the carbon content in a more sensitive range for the backtitration of non-reduced dichromate. The light fraction, consisting mainly of nonhumified higher plant material, constitutes together with surface litter the main food resource of surfaceactive earthworm species such as L. rubellw (Pierce, 19’78). Even where earthworms ingest soil coiltaining humus with radiocarbon dating of several thousand years the worm tissue carbon is of contemporary age
Table 2. Percentage light-fraction organic material (LF), % moisture at -8 kPa and pH (C&I,) of soils used in the experiments, sampled from a lawn, garden, beech forest and spruce forest
UropertY
LF (%) Moisture (%) PH
Soil origin
Lawn
. Garden
3.1 + 0.52 38.7 f 0.59 5.7 rt. 0.11
4.4 + 0.60 46.3 + 0.84 7.1 + 0.01
Soil
_.
Beech forest
Soruce forest
27.7 f 4.28 75.7 i 0.87 4.3 * 0.07
76.1 + 3.01 132.9 F 2.45 3.8 F 0.02
0. DANIELand 1. M. ANDERSON
468
IO
(a ) cl
8
soil cast
lawn
garden
beech
spruce
(b)
(cl
300” 0 al ; 200.-Z E
lawn
7CD
3
2
soil cast
garden
0
1
beech
spruce
soil
garden
lawn
.I! s F
spruce
fe)
(d) 80
Cl soil El
E 0 lil E 2 5:
beech
T
cast
60 2 40 1
20 0
0 lawn
garden
beech
lawn
spruce
garden
beech
spruce
Fig. I. Microbial biomass-C (a), % moisture (b), CO,-C production (c) and organic soluble-c (d) and colony forming units (cfu) of bacteria (e) in four contrasting soils before and after a passage through the gut of the earthworm Lumbricus rubeifus (mean and S.E.).
(Scharpenseel et al., 1989), confirming that LF or litter material with a rapid turnover is the main food resource utihzed by these animals. The higher plant constituents of the LF are in an active phase of decomposition and it is therefore predictable, that the microbial biomass-C in the soils increased with increasing LF content. The microbial biomass-C in the
beech (4.6mg C gg’ soil) and the spruce forest soil (7.5 mg C g-- ’ soil) is similar to that reported by Sparling and West (1988) for peat, humus or litter samples, and corresponds to I .9 and 3.1% of the LF organic-C, respectively. This is about in the range of l-3% total organic C located in microbial biomass found in various temperate and tropical soils (Theng
Table 3. Relative importance of treatment effects with respect to % soil moisture, soluble organic-C, microbial biomass-C, respiration (CO&) and bacterial cfu, as revealed by two-factor analvses of variance Treatment SS as % of total SS
Soil property Microbial biomass-C Moisture (%) CO& Soluble-C Bacterial cfu
Soil origin fd.f. = 3)
Gutpassage (d.f. = 1)
68.9”*
1.4NS
89.3*** x.2*** 42.7’** 41.1***
SS: sum of squares; d.f.: degree '0.01d P ~0.05;NS P > 0.05.
5.5*** 9 o*** 10:5* 24.4.” of
freedom;
Interaction term (d.f. = 3)
2.1NS 1P :;;c: 12.3” ***p 4 0.001;
ErrOr fd.f. = 30)
27.6 4.2 13.2 44.2 22. I **o.ooi Q P 4 0.01;
Microbial biomass and activity in soils: influence of earthworms
et al., 1989). In the garden (2.0mg C g-l) and the lawn soil {0.4mg C g-‘) microbial biomass-C was comparable to the arable and forest soils investigated by Insam (1990) or Martikainen and PalojSirvi (1990), respectively. The higher moisture content in soils with the high LF content, compared to soils with the same water potential and a smaller LF content, is explained by the high water holding capacity of macroorganic matter. Soil respiration also increased with higher LF content, except in the spruce forest soil where the high acidity (pH = 3.8) or chemicals such as phenolic compounds may have suppressed the microbial activity. The difference in soluble organic-C between casts and soil may be the product of several contributory processes. They include selective feeding on nonhumified materials within the mineral soil matrix, digestion by ingested or indigenous enzymes, secretion of mucopolysaccharides by the gut (Arthur, 1963), and incomplete resorption of soluble organicC before the excretion. Similar considerations may apply to the moisture contents in bulk soil and casts. We were unable to distinguish between these mechanisms in our study but tend to rule out selective feeding because of the fine and relatively homogeneous nature of the soil used to feed the worms. Respiration in casts was enhanced compared with the soil, indicating a higher microbial activity. Similar increases in soil respiration after an earthworm gut passage have been reported by Parle (1963) and Scheu (1987a). Conversely, respiration may also be reduced by gut passage of soils amended with a water soluble fraction of grass leaves (Lavelle et al., 1983) but the mechanism was not determined. Respiration measurements after glucose amendment were used to estimate the microbial biomass in earthworm casts of different age (Scheu, 1987a). Soil respiration, however, may change considerably if soil water content, or C-, N- or P-availability changes. Since those factors all are affected by gut passage (Graff, 1970; Loquet, 1978; Scheu, 1987b; Elliot et al., 1990, 1991) and may change during the ageing of the casts, inferences about the microbial biomass in casts drawn from respiratory measurements may be misleading. Since the fumigation-extraction method is a direct measurement of the microbial biomass, it should be a reliable method for estimating the effect of an earthworm gut passage. Our results, which suggest no change in the microbial biomass after a gut passage lasting 6-8 h at 1YC, are consistent with a study by Patra et al. (1990), who showed that annual changes in microbial biomass in an arable and a grassland soil were small and largely masked by experimental and sampling error. If earthworms were to influence the microbial biomass, one would have expected microbial biomass fluctuations due to the seasonality of earthworm activity and the usually large quantities of soil ingested. Many studies (reviewed by Satchell, 1983) have shown higher dilution plate counts of bacteria in casts
469
than in bulk soil and it has generally been assumed, that bacterial numbers are enhanced through gut passage. However, numbers of some Enterobacteriaceae, such as Serratia marcescens, Escherichia coli and Salmonella enteritidis, have also been observed to decrease during gut passage (Day, 1950; Brown and Mitchell, 1981), and Briisewitz (1959) obtained even lower counts in casts compared with soils if glucose was added. The value of dilution plate counts in quantitative ecological studies has been questioned by Buck (1979). The numbers of bacteria growing on a standard nutrient agar are only a small proportion of the totai present and microbial populations are thus substantially underestimated (Herbert, 1982). In addition, those bacteria enumerated using dilution plating are not necessarily those which are functionally important in the ecosystem (Gray, 1990). Hence, higher plate counts in soil after gut passage may be a consequence of an improved dispersion of cells, which were aggregated in plant material or biofilms, to a small portion of the total bacterial community which does proliferate during gut passage, or to the activation of bacteria which are present in the soil in a starvation-survival state (Morita, 1990). Small increases in bacterial numbers, or changes in the activity state, would be unlikely to be detected by measurements of the total microbial biomass, especially against the background of fungi and protozoa (and presumably algae), which also contribute to the microbial biomass (Ingham and Horton, 1987). It is concluded that the passage of soil through earthworm guts changes its physicochemical properties and the level of microbial activity. A change of the microbial biomass, as measured by the fumigation-extraction method, was not detected in 9 h old earthworm casts. The question whether the higher microbial activity after gut passage is due to bacterial cell proliferation, growth of fungal hyphae, or to a metabolic activation of bacterial cells being in a starvation-survival state, has yet to be answered. Acknowledgement-O. Daniel was funded by the Royal Society under the European Science Exchange Programme. REFERENCES
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1992 Copyright
0
0038-0717/92 $5.00 + 0.00 1992 Pergamon Press Ltd
MICROBIAL BIOMASS AND ACTIVITY IN CONTRASTING SOIL MATERIALS AFTER PASSAGE THROUGH THE GUT OF THE EARTHWORM LUMBRICUS RUBELLUS HOFFMEISTER 0. DANIEL’* and J. M. ANDERSON’ I Institute of Terrestrial Ecology/Soil Biology, ETH, CH-8952 Schlieren, Switzerland and *Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Exeter EX4 4PS, U.K. (Accepted
25 November
1991)
Summary-Earthworms (Lumbricus rubehs) were fed on four different soils with light fraction organic material contents from 3.7 to 76.1% of the soil dry weight, and soil water potentials standardized at - 8 kPa. Microbial biomass-C in the soils, as measured with a modified fumigation
INTRODUCTION
MATERIALS AND
METHODS
Soils
In temperate and tropical ecosystems earthworms may ingest large amounts of mineral soil, soil organic matter and surface litter, and produce casts deposited below-ground and on the surface up to as much as 270 t ha-’ yr-t (Lee, 1985). Soil profile development may be influenced by earthworms (Nielson and Hole, 1964), and surface layers of some soils in Europe
(Kubiena, 1953) and African savannas (Lavelle, 1978) may consist almost entirely of earthworm casts. Comparisons between soils and casts indicated that passage through the earthworm gut usually results in higher plate counts of bacteria (Satchell, 1983) and increased rates of microbial respiration (Parle, 1963; Scheu, 1987a). Rates of nitrification (Parle, 1963; Syers et al., 1979; Scheu, 1987b) and denitrification (Elliott et al., 1990, 1991) may also be higher in casts than in bulk soil, indicating that microbial activity is generally stimulated by gut passage. Since microbial biomass is widely used as a key factor mediating soil processes (Jenkinson and Ladd, 1981), we have investigated whether the high microbial activity reported in earthworm casts were related to an increase in the microbial biomass. In order to carry out the measurements the method of Vance et al. (1987) for determining the microbial biomass was callibrated for small soil and cast samples. Four soils with different organic matter content were then used to test the influence of the passage through the gut of Lumbricus rubellus Hoffmeister on the microbial biomass, and related measurements of microbial respiration, bacterial plate counts, soluble organic-C and moisture content.
*Author for correspondence. 465
The soils used were taken from beneath the root mat in a lawn, from a tilled garden plot, and from the humus layer of beech and spruce forests. Aggregates were gently broken down by hand, and stones and roots were removed. The materials were sieved (< 5 mm) and stored in containers at 2-3°C. The soils were prepared for feeding experiments by standardizing moisture contents at - 8 kPa. This involved firming the soil into 4.8 cm dia porous ceramic cups (Soilmoisture Equipment Corp.) to a depth of ca 3 cm, ponding three times with distilled water and allowing equilibrium to be reached with the applied suction tension (80cm water column) over 24 h. The soil moisture content was determined by gravimetry, after drying for 24 h at 105°C. Thereafter, soils stored in containers were carefully moistened with a volume of distilled water sufficient to restore the moisture content to a level equivalent to a water potential of - 8 kPa. The light-fraction (LF) material was separated from the soils by the method of Strickland and Sollins (1987), which was modified for small soil samples. Aliquots of 14 g dry soil (24 h at 105C) were placed in 25 ml universal bottles and dispersed by shaking in I5 ml NaI solution (density = 1.7 g cm-‘) for 1 h on an orbital shaker. The heavy-fraction was allowed to settle for 24 h at room temperature. The LF material on the surface of the NaI solution was transferred by spatula and by aspiration onto a tared, oven-dry glass fibre filter (Whatman GF/C), washed three times by filtration with lOm1 1 M NaCl and three times with distilled water. The weight was then determined after drying 24 h at 105°C. The soil pH was measured in the supernatant of a 1: 2.5 (v: v) soil: 10 mM CaCl, suspension, after shaking and allowing the soil to settle for 3 h.
466
calibration
0. DANIEL and J.M.
of‘ the fumigation-e.~tract~on
inet~lod
The method of Vance et al. (1987) has been used extensively to measure the microbial biomass in a r-angc of soil types (Sparling and West, 1988; Tate (lt ul., 1988; Martikainen and Palojlrvi, 1990; Ocio and Brookes, 1990; Vance and Nadkarni, 1990) and an automated procedure (Wu er al., 1990) has been developed to reduce the time taken for measurements. In our study this method had to be modified for small soil samples such as earthworm casts. Moist soils (equivalent to SO-250 mg dry wt) were weighed into IO ml universal bottles. Non-fumigated soils were extracted immediately, as described below. For the fumigation. the universal bottles were placed in a desiccator lined with wet filter paper to maintain humidity, and containing about 25 ml distilled CHCl, in a flask with anti-bumping granules. The desiccator was evacuated by a vacuum pump until the CHCI, boiled for about 2 min. The evacuated desiccator was then placed in the dark at 25’C. After 24 h the flask with the CHCI, and the filter paper was removed and the desiccator was evacuated 6 times for S min and flushed with air to remove the remaining CHCl, vapour. For the extraction, 4ml 0.5 M K,SO, was added. and the universal bottles were shaken for I h on an orbital shaker. The soil suspension was then filtered through glass fibre filters (Whatman, GF/C) directly into Pyrex 100 ml block digester tubes, with a further two aliquots of 2 ml 0.5 M KzSO, to rinse the universal bottles. Since varying amounts of the extraction solution (depending on soil and quantity) remained in the filters, the volume of the extract in the digester tubes was calculated based on its weight and the density of 0.5 M K?SO, (1.145 g cm-‘). It was assumed that the solution had the same organic-C concentration in the filters and in the digester tubes. The filtered extracts were digested with 10 ml H,SO, (98%), 5 ml H,PO, (SS%), and 2mi 33.3 rnM (fumigated soils) or 16.7 mM (non-fumigated soils) NazCrzO, in a block digester (Kjeldatherm”, Gerhardt GmbH, Bonn) that had been heated to 17O’C. After 30 min the tubes were removed from the block digester, allowed to cool and their contents were quantitatively transferred into 100 ml Erlenmeyer flasks, using distilled H,O to bring the volume to about SO ml. The excess dichromate was determined by backtitration with 16 and 8rn~ ferrous ammonium sulphate in 0.4 M H,SO, for fumigated and respectively. N-phenylannon-fumigated soils, thranilic acid was used as indicator. Losses of dichromate, which are proportional to the amount not used for the oxidation of the organicC in the sample, occur by boiling in a bfock digester; this proportion can be estimated with boiled and unboiled controls (Yeomans and Bremner, 1988). at the low organic-C concentrations However, measured here, constant dichromate losses, due to organic-C from the filters or the chemicals used, may also occur. Since proportional and constant effects
ANDERSON
cannot be separated, a calibration curve with glucose was determined for each digestion run, using 8 concentrations which theoretically used between O-60% of the added dichromate. Microbial biomass-C was calculated as the difference between the organic-C extractable after fumigation and the organic-C extractable without fumigation (soluble-C) multiplied by 2.64 (Vance et al., 1987). This procedure was first validated with the four different soils, determining the extractable-C both with and without fumigation in 5-6 soil portions with dry weights between 50 and 250mg before use on experimental materiais. CO, production CO2 production from casts and soils was measured within 25 ml universal bottles, sealed with SubaSeal bungs, and containing 5 ml 4 mM KOH to absorb the CO: and the sample in a 4mm dia specimen tube attached to the SubaSeal bung. Samples of 20-200 mg dry weight cast or soil were incubated for 48 h in the dark at 25C. The absorbed CO* was then precipitated by 0.4 ml 1 M BaC&, 4 drops of phenolphthalein were added as indicator and the remaining KOH was titrated with 2mM HCl using a microburette. Solutions at working strength were used immediately and at least two blanks were measured per IO samples. Bacterial dilution plate counts Soil and cast samples of 20-200 mg dry wt were dispersed in 10 ml sterile l/4 strength Ringers solution by ultrasonicating at 45 kHz for 4 min, and serial tenfold diluted. One replicate per dilution (0.1 ml) was spread on Oxoid nutrient broth agar plates and incubated for 36-48 h at 2O’C. Zncubation of soils and earth~lor~s To ensure that the animals were in a similar physiological condition for the experiments, L. rubellus was kept for several weeks before the use at 15C in the dark in lawn soil. For the experiments single adult, or subadult, L. rubellus were added to 9 cm dia Petri dishes containing 15-20 g of soil (- 8 kPa water potential) which had been gently compressed to consolidate the structure. Twenty Petri dishes for each of the four soils were placed in covered boxes lined with wet filter paper and kept in the dark at 15°C. After 6-8 h, when the colour of the casts changed except for in lawn soil, all casts were removed from the soil surface. The Petri dishes with the earthworms were incubated a further 16-20 h (i.e. casts had a mean age of 9 h). Soil and casts were sampled then from five Petri dishes each, to compare the microbial biomass-C, organic soluble-C, CO& production, % moisture content and bacteria1 plate counts in casts and bulk soil. Data were transformed with X’ = log(X -t l), where X means microbial biomass-C, organic soluble-C, CO,-C production, % moisture content or bacterial plate counts and X’ is
Microbial
biomass
and activity
467
in soils: influence of earthworms
Table I. Regressions of organic-C (mg gg’) extracted from a range of 6 soil samples (S&250 mg dry wt) before and after chloroform fumigation. Soils were collected from beneath a grass ward (lawn), a tilled garden plot and from beech and spruce forests Extractable-C without fumigation
Lawn Garden Beech forest Spruce forest
usP >
Intercept
Slope
0.003N” O.OlONs 0.042NS 0.044*
0.172*** 0.543*** 0. I 82Ns 0.601***
0.05; ‘P < 0.05; ***p
4
I= 0.801 0.956 0.342 0.860
Extractable-C after fumigation -. 0.067NS 0.017NS -0.042NS - 0.049NS
r2
SlOpe
Intercept
0.570”’ 0.985*** 2.714’** 5.150***
0.568 0.948 0.931 0.992
0.001.
the transformed value, to improve the homogeneity of variances, and the significance of the influence of soil origin and a passage through the gut of L. rubellus was tested by two-factor analyses of variance (Zar, 1984). RESULTS
The modified fumigation-extraction method allowed the determination of extractable organic-C in 50-250mg dry wt soil both before and after CHCI, fumigation. Intercepts of regression lines were below 5Opg C g-’ and, except for the non-fumigated soil from the spruce forest, not significantly different from 0 (Table 1). Slopes of regression lines were significantly different from 0, except for the non-fumigated soil from the beech forest. Coefficients of determination were low for regressions for non-fumigated soil from the beech forest (0.342) and for fumigated soil from the lawn (0.568), but above 0.8 for the other regressions. The LF of the soils increased in the order lawn-garden-beech--spruce over a range from 3.7 to 76.1% of the soil dry weight (Table 2). Assuming that organic matter has 52.6% organic-C (Nelson and Sommers, 1982), the LF organic-C content in the four soils were calculated as respectively 19.5, 23.2, 145.8 and 400.5 mg C g-’ soil. The forest soils with a high LF content were acid, soils from lawn and garden were both in the neutral pH range. Moisture content of the soils at -8 kPa ranged from 38.7 to 132.9%. Microbial biomass-C [Fig. l(a)] and moisture content [Fig. l(b)] in the bulk soil increased with increasing LF content. Microbial biomass-C as % of LF organic-C was 2.1, 8.8, 3.1 and 1.9% in the soils from lawn, garden, and beech and spruce forest, respectively. Soil respiration rate [Fig. l(c)] also increased with increasing LF content, except for the spruce forest soil, where it was lower than in the soil from the beech forest. Amounts of soluble organic-C [Fig.
l(d)] and bacterial plate counts [Fig. l(e)] indicated no relation to the LF content. The origin of the soils had a greater effect on the measured soil properties than a gut passage. This is shown by the relative importance of treatment effects (expressed as treatment SS as % of total SS) which ranged from 41.1 to 89.3% for soil origin and from 1.4 to 24.4% for the gut passage (Table 3). The effect of gut passage on microbial biomass-C was the only treatment which was not significant. DISCUSSIOFi
Microbial biomass measurements by substrate induced respiration (Anderson and Domsch, 1978) fumigation-incubation (Jenkinson and Powlson, 1976) and fumigationextraction (Vance et al., 1987) are usually made on soil samples of 1OOg (wet wt), 250 g (wet wt) and 50 g (dry wt), respectively. Our demonstration that microbiat biomass can be determined on small soil samples of 50-2.50 mg (dry wt) offers the possibility of investigating many processes mediated by invertebrate-microbial interactions operating at this microsite level (Anderson, 1988). Regressions of extractable-C with intercepts being not different from 0 and slopes being different from 0, with one exception each, indicate that the method used is basically sound. For the calibration of nonfumigated soil from the beech forest and for fumigated soil from the lawn, slightly higher soil weights might have been more appropriate to locate the carbon content in a more sensitive range for the backtitration of non-reduced dichromate. The light fraction, consisting mainly of nonhumified higher plant material, constitutes together with surface litter the main food resource of surfaceactive earthworm species such as L. rubellw (Pierce, 19’78). Even where earthworms ingest soil coiltaining humus with radiocarbon dating of several thousand years the worm tissue carbon is of contemporary age
Table 2. Percentage light-fraction organic material (LF), % moisture at -8 kPa and pH (C&I,) of soils used in the experiments, sampled from a lawn, garden, beech forest and spruce forest
UropertY
LF (%) Moisture (%) PH
Soil origin
Lawn
. Garden
3.1 + 0.52 38.7 f 0.59 5.7 rt. 0.11
4.4 + 0.60 46.3 + 0.84 7.1 + 0.01
Soil
_.
Beech forest
Soruce forest
27.7 f 4.28 75.7 i 0.87 4.3 * 0.07
76.1 + 3.01 132.9 F 2.45 3.8 F 0.02
0. DANIELand 1. M. ANDERSON
468
IO
(a ) cl
8
soil cast
lawn
garden
beech
spruce
(b)
(cl
300” 0 al ; 200.-Z E
lawn
7CD
3
2
soil cast
garden
0
1
beech
spruce
soil
garden
lawn
.I! s F
spruce
fe)
(d) 80
Cl soil El
E 0 lil E 2 5:
beech
T
cast
60 2 40 1
20 0
0 lawn
garden
beech
lawn
spruce
garden
beech
spruce
Fig. I. Microbial biomass-C (a), % moisture (b), CO,-C production (c) and organic soluble-c (d) and colony forming units (cfu) of bacteria (e) in four contrasting soils before and after a passage through the gut of the earthworm Lumbricus rubeifus (mean and S.E.).
(Scharpenseel et al., 1989), confirming that LF or litter material with a rapid turnover is the main food resource utihzed by these animals. The higher plant constituents of the LF are in an active phase of decomposition and it is therefore predictable, that the microbial biomass-C in the soils increased with increasing LF content. The microbial biomass-C in the
beech (4.6mg C gg’ soil) and the spruce forest soil (7.5 mg C g-- ’ soil) is similar to that reported by Sparling and West (1988) for peat, humus or litter samples, and corresponds to I .9 and 3.1% of the LF organic-C, respectively. This is about in the range of l-3% total organic C located in microbial biomass found in various temperate and tropical soils (Theng
Table 3. Relative importance of treatment effects with respect to % soil moisture, soluble organic-C, microbial biomass-C, respiration (CO&) and bacterial cfu, as revealed by two-factor analvses of variance Treatment SS as % of total SS
Soil property Microbial biomass-C Moisture (%) CO& Soluble-C Bacterial cfu
Soil origin fd.f. = 3)
Gutpassage (d.f. = 1)
68.9”*
1.4NS
89.3*** x.2*** 42.7’** 41.1***
SS: sum of squares; d.f.: degree '0.01d P ~0.05;NS P > 0.05.
5.5*** 9 o*** 10:5* 24.4.” of
freedom;
Interaction term (d.f. = 3)
2.1NS 1P :;;c: 12.3” ***p 4 0.001;
ErrOr fd.f. = 30)
27.6 4.2 13.2 44.2 22. I **o.ooi Q P 4 0.01;
Microbial biomass and activity in soils: influence of earthworms
et al., 1989). In the garden (2.0mg C g-l) and the lawn soil {0.4mg C g-‘) microbial biomass-C was comparable to the arable and forest soils investigated by Insam (1990) or Martikainen and PalojSirvi (1990), respectively. The higher moisture content in soils with the high LF content, compared to soils with the same water potential and a smaller LF content, is explained by the high water holding capacity of macroorganic matter. Soil respiration also increased with higher LF content, except in the spruce forest soil where the high acidity (pH = 3.8) or chemicals such as phenolic compounds may have suppressed the microbial activity. The difference in soluble organic-C between casts and soil may be the product of several contributory processes. They include selective feeding on nonhumified materials within the mineral soil matrix, digestion by ingested or indigenous enzymes, secretion of mucopolysaccharides by the gut (Arthur, 1963), and incomplete resorption of soluble organicC before the excretion. Similar considerations may apply to the moisture contents in bulk soil and casts. We were unable to distinguish between these mechanisms in our study but tend to rule out selective feeding because of the fine and relatively homogeneous nature of the soil used to feed the worms. Respiration in casts was enhanced compared with the soil, indicating a higher microbial activity. Similar increases in soil respiration after an earthworm gut passage have been reported by Parle (1963) and Scheu (1987a). Conversely, respiration may also be reduced by gut passage of soils amended with a water soluble fraction of grass leaves (Lavelle et al., 1983) but the mechanism was not determined. Respiration measurements after glucose amendment were used to estimate the microbial biomass in earthworm casts of different age (Scheu, 1987a). Soil respiration, however, may change considerably if soil water content, or C-, N- or P-availability changes. Since those factors all are affected by gut passage (Graff, 1970; Loquet, 1978; Scheu, 1987b; Elliot et al., 1990, 1991) and may change during the ageing of the casts, inferences about the microbial biomass in casts drawn from respiratory measurements may be misleading. Since the fumigation-extraction method is a direct measurement of the microbial biomass, it should be a reliable method for estimating the effect of an earthworm gut passage. Our results, which suggest no change in the microbial biomass after a gut passage lasting 6-8 h at 1YC, are consistent with a study by Patra et al. (1990), who showed that annual changes in microbial biomass in an arable and a grassland soil were small and largely masked by experimental and sampling error. If earthworms were to influence the microbial biomass, one would have expected microbial biomass fluctuations due to the seasonality of earthworm activity and the usually large quantities of soil ingested. Many studies (reviewed by Satchell, 1983) have shown higher dilution plate counts of bacteria in casts
469
than in bulk soil and it has generally been assumed, that bacterial numbers are enhanced through gut passage. However, numbers of some Enterobacteriaceae, such as Serratia marcescens, Escherichia coli and Salmonella enteritidis, have also been observed to decrease during gut passage (Day, 1950; Brown and Mitchell, 1981), and Briisewitz (1959) obtained even lower counts in casts compared with soils if glucose was added. The value of dilution plate counts in quantitative ecological studies has been questioned by Buck (1979). The numbers of bacteria growing on a standard nutrient agar are only a small proportion of the totai present and microbial populations are thus substantially underestimated (Herbert, 1982). In addition, those bacteria enumerated using dilution plating are not necessarily those which are functionally important in the ecosystem (Gray, 1990). Hence, higher plate counts in soil after gut passage may be a consequence of an improved dispersion of cells, which were aggregated in plant material or biofilms, to a small portion of the total bacterial community which does proliferate during gut passage, or to the activation of bacteria which are present in the soil in a starvation-survival state (Morita, 1990). Small increases in bacterial numbers, or changes in the activity state, would be unlikely to be detected by measurements of the total microbial biomass, especially against the background of fungi and protozoa (and presumably algae), which also contribute to the microbial biomass (Ingham and Horton, 1987). It is concluded that the passage of soil through earthworm guts changes its physicochemical properties and the level of microbial activity. A change of the microbial biomass, as measured by the fumigation-extraction method, was not detected in 9 h old earthworm casts. The question whether the higher microbial activity after gut passage is due to bacterial cell proliferation, growth of fungal hyphae, or to a metabolic activation of bacterial cells being in a starvation-survival state, has yet to be answered. Acknowledgement-O. Daniel was funded by the Royal Society under the European Science Exchange Programme. REFERENCES
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