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The metabolic quotient for CO2 (qCO2) as a specific activity parameter to assess the effects of environmental conditions, such as ph, on the microbial biomass of forest soils
Soil Biol. Biochem. Vol. 25, No. 3, pp. 393-395, 1993
0038-0717/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SHORT COMMUNICATION
THE METABOLIC QUOTIENT FOR CO, (qC02) AS A SPECIFIC ACTIVITY PARAMETER TO ASSESS THE EFFECTS OF ENVIRONMENTAL CONDITIONS, SUCH AS pH, ON THE MICROBIAL BIOMASS OF FOREST SOILS TRAUTE-HEIDI ANDERSON and K. H. DOMSCH Institut fur Bodenbiologie, BFAL, Bundesallee 50, 3300 Braunschweig, Germany (Accepted
IO September
The metabolic quotient for CO* (qC0,) or specific respiration rate (Pirt, 1975) has been applied to soil microbial biomass analyses in maintenance energy investigations (Anderson and Domsch, 1985a, 1985b), in studies on the effect of temperature (Anderson and Domsch, 1986; Anderson and Gray, 1991; Joergensen et al., 1990), in comparisons of field managements (Anderson and Domsch, 1990), soil variables (Sandtruckova and Straskraba, 1991) in ecosystem successions (Insam and Haselwandter, 1989) and in studies on heavy metal stresses (FlieObach, unpubl. doctoral thesis, University of Giittingen, 1991). In this latter context similar quotients have been used by Brookes and McGrath (1984) and Killham (1985). The usefulness of such quotients for elucidating effects of environmental changes on microbial communities has been demonstrated by Anderson and Gray (1991). This approach was used in a study on the microbial biomass of different forest ecosystems in order to study and quantify the metabolic status of such soils: the use of eco-physiological constants allowed a direct comparison of true microbial activities of different soils. The aim was to test the ecosystem-level hypothesis of “Bioenergetics of Ecosystem Development” put forth by Odum (1969). Our project is part of a long-term research programme on “Stability Criteria of Forest Ecosystems” coordinated by the University of Giittingen. We report on the qC0, of microbial communities of 137 forest stands. With the exception of one stand, all were located in Northern Germany (Lower Saxony). The experiment included comparison between stands with different litter inputs (Fagus and Picea), chronosequences (stands of different ages) and comparison between simple (Fugus, Picea) and mixed (Fugu.s-Quercu.r) forest ecosystems. Soil samples were taken exclusively in early spring (beginning of March) before the emergence of ground-cover. The Oh and Ah (O-5 cm) horizons were sampled separately. Soils were stored unsieued in the cold (4°C) to minimize microbial death during storage. During the following 5 months of experimentation, maximal losses due to storage reached 10% for Ah samples and 25% for Oh samples, when comparing the initial biomass contents of 10 soils (of each horizon) to the biomass contents after 5 months, respectively. From each site, five samples were taken and bulked. Three replicates from the bulk sample were used for microbial biomass or respiration determinations. Before measurements, the soils were sieved ( < 2 mm) and adjusted to a water potential of -240 kpa (range 50-70% w/w for Oh and 30-50% w/w for Ah samples). Soil samples, 10 (Oh) to 25 g (Ah) (dry weight), were used for each analysis, respectively. The substrate-induced respiration (SIR) technique was employed for microbial biomass measurements
1992)
(C,) (Anderson and Domsch, 1978), wherein CO, was analysed with an automated infrared-gas analyser system (Heinemeyer et al., 1989). The amount of glucose applied for obtaining a maximal CO, flush in all soils was 8000 pg g-i. The qC0, (unit CO,-C unit-’ C,, h-i) was determined as described by Anderson and Domsch (1986). CO, evolution from basal respiration (soil without glucose-amendent) was followed until soils had reached a relatively constant CO*production rate at 22°C. This occurred in general after 20 h. The mean of the basal CO* output of the following 10 h was taken for calculating qC0,. Soil pH was determined in 1 M KC1 and soil organic C by dry combustion (Leco). We found that total microbial biomass together with microbial CO,-production rates were far lower (on average 64% for C, or 49% for COr evolution, respectively) in acidic soil conditions than at neutral pH. At the same time, however, with respect to the specific respiration rate (qC0,) of Ah samples the prevalent soil pH strongly al&ted the respiratory activity of soil microorganisms. As demonstrated in Fig. 1 (a, b) for Fugus and Fugw-Quercw forests, respectively, microbial communities released more CO,-C per unit microbial biomass and time under acidic soil conditions than communities at a more neutral pH range. Wolters (1991) also reported an increase in the qC0, of a beech wood (Of layer) following acid-rain treatment. All of the Picea stands had a very low soil pH (range 2.2-3.2; n = 26). Table 1 shows a comparison between qC0, of spruce stands to beech or beech-oak stands all of a similar low pH range (2.7-3.2). In spite of the different ages or substrate qualities of the primary producers, in these low pH environments the metabolic quotients for CO, were very close (qC0, = 2.3 ng pg-i Cd h-i) with the excep tion of the mixed forests (beech-oak) which showed a slightly lower mean qC02 of 2.0 ng pg-’ C, h-l. Unfortunately, at neutral pH conditions, only a few stands of Fugus-Quercus forests have been available to make a useful comparison. Table 1. The metabolic quotient (qC0,) of different forest stands (Ah horizon) at low soil pH (range 2.7-3.2) &0,-C* (ng CO,-?&
C,,h-‘)
mean
Tvnc of forest Fagw
2.35’
Picea
2.3W
SD 0.36 SD 0.40
Fagus-QW~euS
I .9gb
SD 0.40
n = 17 n =19 n =14
PH, mean 3.01 2.90 3.02
*COz production recorded at 22°C.
Values which arc not followed by the same ktter differ significantly at the P < 0.05 level (Student’s r-test); SD = standard deviation.
393
1
1
I
2
3
I
I
A
1
4 PH
d
1
5
1
I
6
I
I
7
i
I
8
Fig. 1. Relationship between microbial CO,-release par unit C, and time and soil pH of (a) Fop stands and (b) Fagus-Quercus stands, respectively; Fugus, n = 69; Fagus-Quercus, n = 34.
0
I
Fogus/Quercus (AI,) A
b)
I
Fagus (AhI
a)
1
f
2.0
0.5
1
2
Fagus/Qucrcus
b)
Fagus (A,,)
3
(A,)
4 PH
A
5
6
l
A
7
l*
0 0
ii
Fig. 2. Relationship between the C,-to-Cp,s ratio and soil pH of (a) Fags stands and (b) Fugus-Quercus stands, respec0vely; Fugus, n = 69; F~gu.+-Qwr~, n=34.
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Short Communications
395
Table 2. The C&-to-C, ratio of different soils (forest and agricultural plots, Ah or Ap horizons, respectively) at low and neutral pH
[%I C,,, ia C,, Type of plot Fagus Pica Fag&-Quercus Agricultural plot@ (NPK-treatment)
PI&, range 2.1-3.2 0.61 0.46 0.61
SD 0.18 SD 0.14 SD 0.20 No data
PI-L, range 5.0-7.9 n = 17 n = 19 n = 14
2.15’ 2.18 2.63
SD 0.73 No data SD 0.53 SD0.40
n = 22 n =2 n=ll
‘Values obtained at neutral pH differ significantly from those obtained at low pH at the P < 0.001 level (Student’s r-test); SD = standard deviation. bAdapted from Anderson and Domsch (1989).
By contrast, the f&-to-C,, ratio (the ratio of microbial carbon to total soil carbon) increased with increasing soil pH [Fig. 2 (a, b)]. A similar observation was reported for some agricultural soils (Kowalczyk and Schrcider, 1988). Interestingly, forest soils at neutral pH showed similar C&-to-C, ratios between 2 and 3%, rather like agricultural soils at comparable soil pH Fable 2). We believe that a high qC0, at low soil pH can be an indicator of terrestrial community stress, which agrees with Odum (1985) who said “repairing damage by disturbances requires diverting energy from growth and production to maintenance”; this is also reflected in our data of microbial biomass per unit C,, at low pH which can differ on average by a factor of 3.5 from microbial biomass at more favorable pH values (Table 2). Data from Oh samples showed a similar trend to that reported for the Ah horizons but with a higher degree of variation. The fact that stresses acting on microbial communities can be quantified by the use of metabolic quotients opens new possibilities in stress ecology. To what extent the higher qC0, values of soils with low pH reflect a higher maintenance energy requirement of the microbial community or indicate a shift in the bacterial-fungal ratio are open questions and will be studied in addition. At the same time, qC0, could be a useful parameter in the study on bioenergetic changes in developing ecosystems (Odum, 1969), where first observations suggested a decrease in the qC0, with progressing maturity of an ecosystem (Insam and Haselwandter, 1989; Anderson and Domsch, 1990). However, the strong influence that pH has on metabolic quotients as such, suggests that the use of the qC0, as a specific activity parameter (or functional index sense Odum, 1969) for the determination of bioenergetic changes of unstressed soil systems should only be applied to soil environments with comparable pH values. Acknowledgements-We are grateful to Professor T. R. G. Gray for helpful comments and linguistic improvements. Special thanks are due to the forest officers, especially Mr Wachter, all of whom helped suggesting forest stands and supplied maps. We thank Mrs Maria Bota for her diligence with the C,, determinations and Mr Kurt Steffens for expert technical assistance. This work was supported by the Bundesministerium filr Forschung und Technologie, Germany.
REFERENCES Anderson J. P. E. and Domsch K. H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215-221. Anderson T.-H. and Domsch K. H. (1985a) Maintenance carbon requirements of actively-metabolizing microbial
populations under in situ conditions. Soil Biology & Biochemistry 17, 197-203. Anderson T.-H. and Domsch K. H. (1985b) Determination of ecophysiological maintenance carbon requirements of soil microorganisms in a dormant state. Biology and Fertility of Soils 1, 81-89. Anderson T.-H. and Domsch K. H. (1986) Carbon assimilation and microbial activity in soil. Zeitschrift fiir P~anzenernaehrung und Bodenkunde 149, 457-468. Anderson T.-H. and Domsch K. H. (1989) Ratios of microbial biomass carbon to total organic carbon in arable soils. Soil Biology & Biochemistry 21, 471-479. Anderson T.-H. and Domsch K. H. (1990) Application of eco-physiological quotients (qC0, and qD) on microbial biomasses from soils of different cropping histories. Soil Biology & Biochemistry 22, 251-255: _ Anderson T.-H. and Grav T. R. G. (19911 The influence of soil organic carbon on microbial growth and survival. In Advances in Soil Organic Matter Research: The Impact On Agriculture & The Environment (W. S. Wilson, Ed.), pp. 253-266. Redwood Press, Melksham. Brookes P. C. and McGrath S. P. (1984) Effects of metal toxicity on the size of the soil microbial biomass. Journal of Soil Science 35, 341-356. Heinemeyer O., Insam H., Kaiser E.-A. and Walenzik G. (1989) Soil microbial biomass and respiration measurements: an automated technique based on infra-red gas analysis. Plant and Soil 116, 191-195. Insam H. and Haselwandter K. (1989) Metabolic quotient of the soil microflora in relation to plant succession. Oecologia 79, 171-178. Joergensen R. G., Brookes P. C. and Jenkinson D. S. (1990) Survival of the soil microbial biomass at elevated temperatures. Soil Biology & Biochemistry 22, 1129-l 136. Killham K. (1985) A physiological determination of the impact of environmental stress on the activity of microbial biomass. Environmental Pollution 38, 283-294. Kowalczyk T. and Schroder D. (1988) Beeinflussung bodenmikrobiologischer Parameter durch Bodeneigenschaften auf Standorten mit geringen Unterschieden im C,,-Gehalt. Kali-Briefe (Biintehofl 19, 335-344. Odum E. (1969) The strategy of ecosystem development. Science 164, 262-270. Odum E. (1985) Trends expected in stressed ecosystems. Bioscience 35, 419-422. Pirt S. J. (1975) Principles of Microbe and Cell Cultivation. Blackwell, Oxford. Santruckova H. and Straskraba M. (1991) On the relationship between specific respiration activity and microbial biomass in soils. Soil Biology & Biochemistry 23, 525-532. Wolters V. (1991) Biological processes in two beech forest soils treated with simulated acid rain-a laboratory experiment with Isotoma tigrina (Insecta, Collembola). Soil Biology & Biochemistry 23, 381-390.
0038-0717/93 $6.00 + 0.00 Copyright 0 1993 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
SHORT COMMUNICATION
THE METABOLIC QUOTIENT FOR CO, (qC02) AS A SPECIFIC ACTIVITY PARAMETER TO ASSESS THE EFFECTS OF ENVIRONMENTAL CONDITIONS, SUCH AS pH, ON THE MICROBIAL BIOMASS OF FOREST SOILS TRAUTE-HEIDI ANDERSON and K. H. DOMSCH Institut fur Bodenbiologie, BFAL, Bundesallee 50, 3300 Braunschweig, Germany (Accepted
IO September
The metabolic quotient for CO* (qC0,) or specific respiration rate (Pirt, 1975) has been applied to soil microbial biomass analyses in maintenance energy investigations (Anderson and Domsch, 1985a, 1985b), in studies on the effect of temperature (Anderson and Domsch, 1986; Anderson and Gray, 1991; Joergensen et al., 1990), in comparisons of field managements (Anderson and Domsch, 1990), soil variables (Sandtruckova and Straskraba, 1991) in ecosystem successions (Insam and Haselwandter, 1989) and in studies on heavy metal stresses (FlieObach, unpubl. doctoral thesis, University of Giittingen, 1991). In this latter context similar quotients have been used by Brookes and McGrath (1984) and Killham (1985). The usefulness of such quotients for elucidating effects of environmental changes on microbial communities has been demonstrated by Anderson and Gray (1991). This approach was used in a study on the microbial biomass of different forest ecosystems in order to study and quantify the metabolic status of such soils: the use of eco-physiological constants allowed a direct comparison of true microbial activities of different soils. The aim was to test the ecosystem-level hypothesis of “Bioenergetics of Ecosystem Development” put forth by Odum (1969). Our project is part of a long-term research programme on “Stability Criteria of Forest Ecosystems” coordinated by the University of Giittingen. We report on the qC0, of microbial communities of 137 forest stands. With the exception of one stand, all were located in Northern Germany (Lower Saxony). The experiment included comparison between stands with different litter inputs (Fagus and Picea), chronosequences (stands of different ages) and comparison between simple (Fugus, Picea) and mixed (Fugu.s-Quercu.r) forest ecosystems. Soil samples were taken exclusively in early spring (beginning of March) before the emergence of ground-cover. The Oh and Ah (O-5 cm) horizons were sampled separately. Soils were stored unsieued in the cold (4°C) to minimize microbial death during storage. During the following 5 months of experimentation, maximal losses due to storage reached 10% for Ah samples and 25% for Oh samples, when comparing the initial biomass contents of 10 soils (of each horizon) to the biomass contents after 5 months, respectively. From each site, five samples were taken and bulked. Three replicates from the bulk sample were used for microbial biomass or respiration determinations. Before measurements, the soils were sieved ( < 2 mm) and adjusted to a water potential of -240 kpa (range 50-70% w/w for Oh and 30-50% w/w for Ah samples). Soil samples, 10 (Oh) to 25 g (Ah) (dry weight), were used for each analysis, respectively. The substrate-induced respiration (SIR) technique was employed for microbial biomass measurements
1992)
(C,) (Anderson and Domsch, 1978), wherein CO, was analysed with an automated infrared-gas analyser system (Heinemeyer et al., 1989). The amount of glucose applied for obtaining a maximal CO, flush in all soils was 8000 pg g-i. The qC0, (unit CO,-C unit-’ C,, h-i) was determined as described by Anderson and Domsch (1986). CO, evolution from basal respiration (soil without glucose-amendent) was followed until soils had reached a relatively constant CO*production rate at 22°C. This occurred in general after 20 h. The mean of the basal CO* output of the following 10 h was taken for calculating qC0,. Soil pH was determined in 1 M KC1 and soil organic C by dry combustion (Leco). We found that total microbial biomass together with microbial CO,-production rates were far lower (on average 64% for C, or 49% for COr evolution, respectively) in acidic soil conditions than at neutral pH. At the same time, however, with respect to the specific respiration rate (qC0,) of Ah samples the prevalent soil pH strongly al&ted the respiratory activity of soil microorganisms. As demonstrated in Fig. 1 (a, b) for Fugus and Fugw-Quercw forests, respectively, microbial communities released more CO,-C per unit microbial biomass and time under acidic soil conditions than communities at a more neutral pH range. Wolters (1991) also reported an increase in the qC0, of a beech wood (Of layer) following acid-rain treatment. All of the Picea stands had a very low soil pH (range 2.2-3.2; n = 26). Table 1 shows a comparison between qC0, of spruce stands to beech or beech-oak stands all of a similar low pH range (2.7-3.2). In spite of the different ages or substrate qualities of the primary producers, in these low pH environments the metabolic quotients for CO, were very close (qC0, = 2.3 ng pg-i Cd h-i) with the excep tion of the mixed forests (beech-oak) which showed a slightly lower mean qC02 of 2.0 ng pg-’ C, h-l. Unfortunately, at neutral pH conditions, only a few stands of Fugus-Quercus forests have been available to make a useful comparison. Table 1. The metabolic quotient (qC0,) of different forest stands (Ah horizon) at low soil pH (range 2.7-3.2) &0,-C* (ng CO,-?&
C,,h-‘)
mean
Tvnc of forest Fagw
2.35’
Picea
2.3W
SD 0.36 SD 0.40
Fagus-QW~euS
I .9gb
SD 0.40
n = 17 n =19 n =14
PH, mean 3.01 2.90 3.02
*COz production recorded at 22°C.
Values which arc not followed by the same ktter differ significantly at the P < 0.05 level (Student’s r-test); SD = standard deviation.
393
1
1
I
2
3
I
I
A
1
4 PH
d
1
5
1
I
6
I
I
7
i
I
8
Fig. 1. Relationship between microbial CO,-release par unit C, and time and soil pH of (a) Fop stands and (b) Fagus-Quercus stands, respectively; Fugus, n = 69; Fagus-Quercus, n = 34.
0
I
Fogus/Quercus (AI,) A
b)
I
Fagus (AhI
a)
1
f
2.0
0.5
1
2
Fagus/Qucrcus
b)
Fagus (A,,)
3
(A,)
4 PH
A
5
6
l
A
7
l*
0 0
ii
Fig. 2. Relationship between the C,-to-Cp,s ratio and soil pH of (a) Fags stands and (b) Fugus-Quercus stands, respec0vely; Fugus, n = 69; F~gu.+-Qwr~, n=34.
E t
$ 1.0 co
*ff 1.5 v
$
v
g 2.5
3.0
0
*
j.-
Lo If
3
vc B E
4
Short Communications
395
Table 2. The C&-to-C, ratio of different soils (forest and agricultural plots, Ah or Ap horizons, respectively) at low and neutral pH
[%I C,,, ia C,, Type of plot Fagus Pica Fag&-Quercus Agricultural plot@ (NPK-treatment)
PI&, range 2.1-3.2 0.61 0.46 0.61
SD 0.18 SD 0.14 SD 0.20 No data
PI-L, range 5.0-7.9 n = 17 n = 19 n = 14
2.15’ 2.18 2.63
SD 0.73 No data SD 0.53 SD0.40
n = 22 n =2 n=ll
‘Values obtained at neutral pH differ significantly from those obtained at low pH at the P < 0.001 level (Student’s r-test); SD = standard deviation. bAdapted from Anderson and Domsch (1989).
By contrast, the f&-to-C,, ratio (the ratio of microbial carbon to total soil carbon) increased with increasing soil pH [Fig. 2 (a, b)]. A similar observation was reported for some agricultural soils (Kowalczyk and Schrcider, 1988). Interestingly, forest soils at neutral pH showed similar C&-to-C, ratios between 2 and 3%, rather like agricultural soils at comparable soil pH Fable 2). We believe that a high qC0, at low soil pH can be an indicator of terrestrial community stress, which agrees with Odum (1985) who said “repairing damage by disturbances requires diverting energy from growth and production to maintenance”; this is also reflected in our data of microbial biomass per unit C,, at low pH which can differ on average by a factor of 3.5 from microbial biomass at more favorable pH values (Table 2). Data from Oh samples showed a similar trend to that reported for the Ah horizons but with a higher degree of variation. The fact that stresses acting on microbial communities can be quantified by the use of metabolic quotients opens new possibilities in stress ecology. To what extent the higher qC0, values of soils with low pH reflect a higher maintenance energy requirement of the microbial community or indicate a shift in the bacterial-fungal ratio are open questions and will be studied in addition. At the same time, qC0, could be a useful parameter in the study on bioenergetic changes in developing ecosystems (Odum, 1969), where first observations suggested a decrease in the qC0, with progressing maturity of an ecosystem (Insam and Haselwandter, 1989; Anderson and Domsch, 1990). However, the strong influence that pH has on metabolic quotients as such, suggests that the use of the qC0, as a specific activity parameter (or functional index sense Odum, 1969) for the determination of bioenergetic changes of unstressed soil systems should only be applied to soil environments with comparable pH values. Acknowledgements-We are grateful to Professor T. R. G. Gray for helpful comments and linguistic improvements. Special thanks are due to the forest officers, especially Mr Wachter, all of whom helped suggesting forest stands and supplied maps. We thank Mrs Maria Bota for her diligence with the C,, determinations and Mr Kurt Steffens for expert technical assistance. This work was supported by the Bundesministerium filr Forschung und Technologie, Germany.
REFERENCES Anderson J. P. E. and Domsch K. H. (1978) A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biology & Biochemistry 10, 215-221. Anderson T.-H. and Domsch K. H. (1985a) Maintenance carbon requirements of actively-metabolizing microbial
populations under in situ conditions. Soil Biology & Biochemistry 17, 197-203. Anderson T.-H. and Domsch K. H. (1985b) Determination of ecophysiological maintenance carbon requirements of soil microorganisms in a dormant state. Biology and Fertility of Soils 1, 81-89. Anderson T.-H. and Domsch K. H. (1986) Carbon assimilation and microbial activity in soil. Zeitschrift fiir P~anzenernaehrung und Bodenkunde 149, 457-468. Anderson T.-H. and Domsch K. H. (1989) Ratios of microbial biomass carbon to total organic carbon in arable soils. Soil Biology & Biochemistry 21, 471-479. Anderson T.-H. and Domsch K. H. (1990) Application of eco-physiological quotients (qC0, and qD) on microbial biomasses from soils of different cropping histories. Soil Biology & Biochemistry 22, 251-255: _ Anderson T.-H. and Grav T. R. G. (19911 The influence of soil organic carbon on microbial growth and survival. In Advances in Soil Organic Matter Research: The Impact On Agriculture & The Environment (W. S. Wilson, Ed.), pp. 253-266. Redwood Press, Melksham. Brookes P. C. and McGrath S. P. (1984) Effects of metal toxicity on the size of the soil microbial biomass. Journal of Soil Science 35, 341-356. Heinemeyer O., Insam H., Kaiser E.-A. and Walenzik G. (1989) Soil microbial biomass and respiration measurements: an automated technique based on infra-red gas analysis. Plant and Soil 116, 191-195. Insam H. and Haselwandter K. (1989) Metabolic quotient of the soil microflora in relation to plant succession. Oecologia 79, 171-178. Joergensen R. G., Brookes P. C. and Jenkinson D. S. (1990) Survival of the soil microbial biomass at elevated temperatures. Soil Biology & Biochemistry 22, 1129-l 136. Killham K. (1985) A physiological determination of the impact of environmental stress on the activity of microbial biomass. Environmental Pollution 38, 283-294. Kowalczyk T. and Schroder D. (1988) Beeinflussung bodenmikrobiologischer Parameter durch Bodeneigenschaften auf Standorten mit geringen Unterschieden im C,,-Gehalt. Kali-Briefe (Biintehofl 19, 335-344. Odum E. (1969) The strategy of ecosystem development. Science 164, 262-270. Odum E. (1985) Trends expected in stressed ecosystems. Bioscience 35, 419-422. Pirt S. J. (1975) Principles of Microbe and Cell Cultivation. Blackwell, Oxford. Santruckova H. and Straskraba M. (1991) On the relationship between specific respiration activity and microbial biomass in soils. Soil Biology & Biochemistry 23, 525-532. Wolters V. (1991) Biological processes in two beech forest soils treated with simulated acid rain-a laboratory experiment with Isotoma tigrina (Insecta, Collembola). Soil Biology & Biochemistry 23, 381-390.