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The effect of artificial acid rain on respiration and cellulase activity in Norway spruce needle litter
003X-0717 8s OlOl-~23SO~.~O
Soii B&f. Eiochem. Vol. 13, pp. 23 to 26 0 Pergamon Press Lid 1981. Prinled in Great Bntain
THE EFFECT OF ARTIFICIAL ACID RAIN ON RESPIRATION AND CELLULASE ACTIVITY IN NORWAY SPRUCE NEEDLE LITTER J. HOVLAND* Department of Microbiology, A~icultural University of Norway, N- 1432 Aas-NLH, Norway (Accepted
15 July
1980)
Summary-Treatment of Norway spruce forest with artificial acid rain at extreme pH values decreased the pH of the needle litter. Extractable cellulase activity showed a low correlation with litter pH and none with moisture. The respiration was correlated with a second degree polynomial of the moisture. When cellulase was included in the multiple regression, with respiration as the dependent variable, the correlation was improved. It is indicated that acid precipitation has a small effect on biologicaf activity in the litter measured as respiration and cellulase.
Emissions of sulphur dioxide are oxidized in the air, forming sulphuric acid. The pH of the precipitation has been reduced from earlier values above 5.0 to annual averages between 4.0 and 5.0 in several areas of Europe (OECD, 1977). Some authors have postulated that acid precipitation decreases the rate of forest litter de~mposition (Oden, 1968; Malmer, 1974; Tamm et al., 1977). The respiration can be used as a measure of the total biological activity in soil. It is possible that ccllulase activity may be a more effective indicator of needle litter decomposition as cellulose forms a substantial part of the litter. Enzymatic measurements in forest soils and needle litter have been used to investigate the effect of heavy metals (Tyler, 1976) and biocida1 treatments on decomposition (Spalding, 1978). My aim was to investigate the influence of artificial acid rain on soil respiration and cellulase activity. MATERIALS
AND METHODS
Study area and sampling The experimental field A-2 at Nordmoen, county of Akershus, of the SNSF-project was used (Abrahamsen et al., 1976a; Stuanes and Sveistrup, 1979). The field was clear-cut and planted with Norway spruce (Piceu abies (L.) Karst.) in 1956. The plantation is divided into f2 plots (3 repli~tions) which have been watered with groundwater or groundwater with H2S04, in addition to normal rain, since June 1973. The water used for the four treatments has a pH of 6.1, 4.0, 3.0 or 2.5 respectively. The waterings are carried out once a month during the frost-free period of the year in a amounts equal to 50 mm of pr~~pitation. At the time of sampling 27 waterings had been carried out, the last one before sampling in the second week of Sep tember 1978.
*Present address: Norsk Hydro its, Research Centre, N-3901 Porsgrunn, Norway.
Litter samples were collected 18 October 1978. The litter layer consisting of spruce needles was very shallow and only located beneath the trees. On each plot, samples ware taken from beneath 3 trees. The samples were taken in triplicate by means of a cork borer (25 mm dia). These 3 samples were then pooled. The samples sites were selected to minimize litter other than spruce needles. Any green parts and roots of the ground vegetation were removed. The thickness of the samples were approximately 1 cm and consisted of the 0,. and OF layers. Due to frequent rain during the last weeks before sampling the litter was moist. Good conditions for fungal growth were indicated by the presence of litter decomposing Basidiomycetes, mainly of the genera My~e~a and Murasmius. The samples were put into polyethylene bottles and brought to the laboratory. Analytical methods
Carbon dioxide evolution was measured at 21“C by pumping CO,-free air through a washing bottle containing water and then through a glass-tube containing the sample. The air flow was measured by means of a rotameter and the C02-concentration was measured with an infra-red U&.-analyzer (Mod. 225, The Analytical Development Co., Hoddesdon, England), and dry matter (DM) was determined on a subsample (1OYC). An extract for the measurement of celiulase activity was prepared by grinding I g of a moist sample with 9 ml of 0.1 M sodium borate buffer, pH 7 (GoksByr and Eriksen, personal communication) to a paste in a mortar and further homogenized with an Ilado X-1020 Mixer for 1 min. The suspension was centtifuged and the supernatant filtered through a Whatman GF/C glass-fibre filter and a 0.45 pm membrane filter. Sodium azide (0.02%) was added as a preservative. This extract could be stored for several days without a reduction of the activity. Cellulase activity was measured by viscometry as described by Almin and Eriksson (1967) and Almin rt al. (1967). A Cannon-Fenske Viscometer No. 300 was 23
24
J. HOVLAND Table
1. Linear
correlation
Respiration
Cellulase
0.42’ 0.22 0.03 0.40*
Cellulase “Rain” pH Litter pH(S) Moisture * Significant t Significant
coefficients variables
pH
measured
Litter pH(S)
0.36t 0.10
0.53*
at 1% level. at 5% level.
3.0
4.0 pH of artificial
between
“Rain”
0.04 oso* -0.10
used. A solution of 0.5% carboxymethylcellulose (Fluka) in 50mM sodium acetate buffer, pH 5, was used as the enzyme substrate. One ml of extract was added to 20ml CMC-solution. The measurements were made at 30°C. The rate of efflux was measured every 5 min for 30 min. A regression line was fitted to the inverse specific viscosity as a function of reaction time. The cellulase activity was calculated as relative units in accordance with equation 6 of Almin and Eriksson (1967). The pH of the moist litter was measured by pressing a surface pH-electrode (Lot 403-30-M3, Ingold, Frankfurt) onto the compressed litter sampled (pH(S)) and by suspending 1 g of moist litter in 3 ml distilled water (pH(H,O)).
6.1
(r)
2.5
rain
‘Fig. 1. The pH(S) of the spruce needle litter treated artificial rain of different pH values.
with
RESULTS AND DISCUSSION
In Fig. 1 are shown the pH in the litter due to the application of acidified water. The decrease in pH was significant (Table l), and is in agreement with the results of Abrahamsen et al. (1976b). There was also a decrease in litter pH with increasing natural moisture content of the litter samples (Fig. 2, Table 1). This relationship was independent of the method used for the pH measurements. Measurements with the surface electrode gave results averaging 0.78 units lower than measurements with the litter suspended in water. The extractable amount of cellulase was significantly less in the samples with a low pH (Fig. 3, Table 1). Approximately 25% of the variation in cellulase was explained by litter pH(S). The pH(H20) data are not used since they are closely correlated with the pH(S) data. Usually fungal cellulases have pH optima at pH 4.5-5.0. Possibly the in situ activity would be even less in the litter samples with a low pH compared to those with a pH approximately 4.5. Previous investigations with acidification of soils (Smith and Whitehead, 1940; White et al., 1949; Schmidt and Ruschmeyer, 1958; Ruschmeyer and Schmidt, 1958) have in some cases shown a decreased cellulolytic activity with decreasing pH. However, some of these experiments on the effect of pH have been carried out using different soils with different pHs. This makes it difficult to come to a conclusion on the effect of pH alone as also other factors varies.
6.0 0 0
t
z .-
z I a
. I
I
I
50
100
150
Moisture Fig. 2. Relationship
between
. T 200
(% of DM)
the pH and the moisture
of the litter.
l pH(S); 0 pH(H20).
Acid rain and litter decomposition
1500
In a comparison of the respiration in the organic layer of different soils, the variation in the respiration could not be explained by the variation in pH (Jorgensen and Wells, 1973). The correlation between moisture and respiration could be significantly improved by using a second degree polynomial. The linear correlation coefficient squared was 0.16 while the coefficient for the polynomial was 0.31 (Table 2). For this reason the moisture squared was included in the multiple regressions, with respiration as the dependent variable. Moisture and pH together explained 37% of the variation in the respiration measurements, and less than 6% was explained by a variation in litter pH(S) between 3.5 and 4.8. The optimum moisture for respiration was approximately 140% of DM (Fig. 4) with a respiration value higher than 90% of maximum between 100 and 180% moisture, when calculated from the fitted equation. However, it does not seem justified to conclude that the respiration was reduced above 140% moisture as there are few measurements. This is in agreement with Heal et al. (1978) who found that the respiration was inhibited by moisture levels below
.
. ‘g 1000 0, ui x s . 8 i
500
&
4.0 Litter pH(S)
3.5
4.5
Fig. 3. Relationship between the cellulase activity and the
pH(S) of the litter.
0
1
25
I
I
I
I
50
100
150
200
Moisture
(% of DM )
Fig. 4. Relationship between the respiration and the moisture of the litter.
Table 2. Correlation coefficient in multiple regressions with cellulase and respiration as dependent variables Dependent variable Cellulase Respiration
Respiration
Step No.
Correlation coefficient, RZ
Variables in regression
1 2
Litter pH(S) Litter pH(S)
Moisture
0.25 0.25
1
Moisture Moisture Moisture
Moisture2 Moisture’
Litter pH(S)
2 3 1 2 3 4
Cellulase Cellulase Cellulase Cellulase
Moisture Moisture Moisture
Moisture’ Moisture’
0.38 0.31 0.16 Litter pH(S)
0.63 0.61 0.46 0.18
J. HOVLAND
26
1000
500 Cellulase
(units
. g DM-’
1500
)
Fig. 5. Relationship between the respiration and the cellulase activity of the litter. lOO%, but appeared to be independent of moisture up to 600%. Spalding (1977, 1978) has shown cellulase activity in coniferous litter to be highly correlated with respiration. The present investigation displays a similar conclusion (Fig. 5, Table 1) when cellulase was included among the variables explaining the variation in respiration, the correlation coefficient was increased considerably (Table 2). My results seem to indicate that under natural conditions the reduced pH in coniferous needle litter due to acid rain may be of small importance for the biological activity measured as cellulase activity and respiration. Acknowledgement-I thank Mr Erik Mmness, The Computing Centre of the Agricultural University, for his assistante with the statistical computations.
REFERENCES ABRAHAMSEN G., BJOR K. and TEIGEN 0. (1976a) Field experiments with simulated acid precipitation in forest ecosystems-1. Soil and vegetation characteristics, experimental design and equipment. SNSF-project FR 4/76, Oslo and As. ABRAHAMSEN G., BJOR K., HORNTVEDT R. and TVEITE B. (1976b) Effects of acid precipitation on coniferous forest. In Impact of Acid Precipitation on Forest and Freshwater Ecosystems in Norway (F. H. Braekke, Ed.), pp. 37-63. SNSF-project FR 6/76, Oslo and As.
ALMIN K. E. and ERIKSSON K.-E. (1967) Enzymic degradation of polymers-I. Viscometric method for the determination of enzymic activity. Biochimica et Biophysicu Actu 139, 238-247. ALMIN K. E.. ERIKSSON K.-E. and JANSSON C. (1967) Enzymic degradation of polymers--II. Viscometric determination of cellulase activity in absolute terms. Biochimicu et Biophysics Acta 139, 248-253. HEAL 0. W., LATTER P. M. and HOW~ON G. (1978) A study of the rates of decomposition of organic matter. In Ecolouical Studies. Vol. 27 (0. W. Heal and D. F. Perkins. E&). pp. 136-161. Springer. Berlin. JORGENSENJ. R. and WELLS C. G. (1973) The relationship of respiration in organic and mineral soil layers to soil chemical properties. PIant & Soil 39, 373-387. MALMER N. (1974) On the effects on water, soil and vegetation of an increasing atmospheric supply of sulphur. A survey on ecological bases. Statens naturvirdssverk SNV PM 402 E, Stockholm. ODEN S. (1968) NederbBrdens och luftens fiirsurning-dess orsaker. fiirlopp och verkan i olika miljiier. Ecoloyical Rrseurch Committee Bulletin. Stockholm 1, l-87. OECD (1977) The OECD programme on long range transport of air pollutants. Measurements and findings. Organisation for economic Co-operation and Development, Paris. RUSCHMEYER 0. R. and SCHMIDT E. L. (1958) Cellulose decomposition in soil burial beds-II. Cellulolytic activity as influenced by alteration of soil properties. Applied Microbiology 6, 115-I 20. SCHMIDT E. L. and RUSCHMEYER 0. R. (1958) Cellulose decomposition in soil burial beds-I. Soil properties in relation to cellulose degradation. Applied Microbiology 6, 108&l 14. SMITH F. B. and WHITEHEAD T. JR (1940) The Effect of substituted cations in the soil complex on the decomposition of organic matter. Proceedings. Soil Science Society of America 5, 248-253. SPALDING B. P. (1977) Enzvmatic activities related to the decomposition of conife;ous leaf litter. Soil Science Society of America Journal 41, 622-627. SPALDING B. P. (1978) The effect of biocidal treatments on respiration and enzymatic activities of Douglas-fir needle litter. Soil Biology d; Biochemistry 10, 537-T43. STUANES A. and SVEISTRUP T. E. (1979) Field experiments with simulated acid rain in forest ecosystems-II. Description and classification of the soils used in field, lysimeter and laboratory experiments. SNSF Project FR 15179, Oslo and As. TAMM C. O., WIKLANDER G. and POPOVIC B. (1977) Effects of application of acid to poor pine forest. Water, Air and Soil Pollution 8, 75-87. TYLER G. (1976) Heavy metal pollution. phosphatase activity, and mineralization of organic phosphorus in forest soils. Soil Biology & Biochemistry 8, 327-332. WHITE J. W.. HOLBECH F. J. and JEFFRIES C. D. (1949) Cellulose-decomposing power in relation to reaction of soils. Soil Science 68, 229-235.
Soii B&f. Eiochem. Vol. 13, pp. 23 to 26 0 Pergamon Press Lid 1981. Prinled in Great Bntain
THE EFFECT OF ARTIFICIAL ACID RAIN ON RESPIRATION AND CELLULASE ACTIVITY IN NORWAY SPRUCE NEEDLE LITTER J. HOVLAND* Department of Microbiology, A~icultural University of Norway, N- 1432 Aas-NLH, Norway (Accepted
15 July
1980)
Summary-Treatment of Norway spruce forest with artificial acid rain at extreme pH values decreased the pH of the needle litter. Extractable cellulase activity showed a low correlation with litter pH and none with moisture. The respiration was correlated with a second degree polynomial of the moisture. When cellulase was included in the multiple regression, with respiration as the dependent variable, the correlation was improved. It is indicated that acid precipitation has a small effect on biologicaf activity in the litter measured as respiration and cellulase.
Emissions of sulphur dioxide are oxidized in the air, forming sulphuric acid. The pH of the precipitation has been reduced from earlier values above 5.0 to annual averages between 4.0 and 5.0 in several areas of Europe (OECD, 1977). Some authors have postulated that acid precipitation decreases the rate of forest litter de~mposition (Oden, 1968; Malmer, 1974; Tamm et al., 1977). The respiration can be used as a measure of the total biological activity in soil. It is possible that ccllulase activity may be a more effective indicator of needle litter decomposition as cellulose forms a substantial part of the litter. Enzymatic measurements in forest soils and needle litter have been used to investigate the effect of heavy metals (Tyler, 1976) and biocida1 treatments on decomposition (Spalding, 1978). My aim was to investigate the influence of artificial acid rain on soil respiration and cellulase activity. MATERIALS
AND METHODS
Study area and sampling The experimental field A-2 at Nordmoen, county of Akershus, of the SNSF-project was used (Abrahamsen et al., 1976a; Stuanes and Sveistrup, 1979). The field was clear-cut and planted with Norway spruce (Piceu abies (L.) Karst.) in 1956. The plantation is divided into f2 plots (3 repli~tions) which have been watered with groundwater or groundwater with H2S04, in addition to normal rain, since June 1973. The water used for the four treatments has a pH of 6.1, 4.0, 3.0 or 2.5 respectively. The waterings are carried out once a month during the frost-free period of the year in a amounts equal to 50 mm of pr~~pitation. At the time of sampling 27 waterings had been carried out, the last one before sampling in the second week of Sep tember 1978.
*Present address: Norsk Hydro its, Research Centre, N-3901 Porsgrunn, Norway.
Litter samples were collected 18 October 1978. The litter layer consisting of spruce needles was very shallow and only located beneath the trees. On each plot, samples ware taken from beneath 3 trees. The samples were taken in triplicate by means of a cork borer (25 mm dia). These 3 samples were then pooled. The samples sites were selected to minimize litter other than spruce needles. Any green parts and roots of the ground vegetation were removed. The thickness of the samples were approximately 1 cm and consisted of the 0,. and OF layers. Due to frequent rain during the last weeks before sampling the litter was moist. Good conditions for fungal growth were indicated by the presence of litter decomposing Basidiomycetes, mainly of the genera My~e~a and Murasmius. The samples were put into polyethylene bottles and brought to the laboratory. Analytical methods
Carbon dioxide evolution was measured at 21“C by pumping CO,-free air through a washing bottle containing water and then through a glass-tube containing the sample. The air flow was measured by means of a rotameter and the C02-concentration was measured with an infra-red U&.-analyzer (Mod. 225, The Analytical Development Co., Hoddesdon, England), and dry matter (DM) was determined on a subsample (1OYC). An extract for the measurement of celiulase activity was prepared by grinding I g of a moist sample with 9 ml of 0.1 M sodium borate buffer, pH 7 (GoksByr and Eriksen, personal communication) to a paste in a mortar and further homogenized with an Ilado X-1020 Mixer for 1 min. The suspension was centtifuged and the supernatant filtered through a Whatman GF/C glass-fibre filter and a 0.45 pm membrane filter. Sodium azide (0.02%) was added as a preservative. This extract could be stored for several days without a reduction of the activity. Cellulase activity was measured by viscometry as described by Almin and Eriksson (1967) and Almin rt al. (1967). A Cannon-Fenske Viscometer No. 300 was 23
24
J. HOVLAND Table
1. Linear
correlation
Respiration
Cellulase
0.42’ 0.22 0.03 0.40*
Cellulase “Rain” pH Litter pH(S) Moisture * Significant t Significant
coefficients variables
pH
measured
Litter pH(S)
0.36t 0.10
0.53*
at 1% level. at 5% level.
3.0
4.0 pH of artificial
between
“Rain”
0.04 oso* -0.10
used. A solution of 0.5% carboxymethylcellulose (Fluka) in 50mM sodium acetate buffer, pH 5, was used as the enzyme substrate. One ml of extract was added to 20ml CMC-solution. The measurements were made at 30°C. The rate of efflux was measured every 5 min for 30 min. A regression line was fitted to the inverse specific viscosity as a function of reaction time. The cellulase activity was calculated as relative units in accordance with equation 6 of Almin and Eriksson (1967). The pH of the moist litter was measured by pressing a surface pH-electrode (Lot 403-30-M3, Ingold, Frankfurt) onto the compressed litter sampled (pH(S)) and by suspending 1 g of moist litter in 3 ml distilled water (pH(H,O)).
6.1
(r)
2.5
rain
‘Fig. 1. The pH(S) of the spruce needle litter treated artificial rain of different pH values.
with
RESULTS AND DISCUSSION
In Fig. 1 are shown the pH in the litter due to the application of acidified water. The decrease in pH was significant (Table l), and is in agreement with the results of Abrahamsen et al. (1976b). There was also a decrease in litter pH with increasing natural moisture content of the litter samples (Fig. 2, Table 1). This relationship was independent of the method used for the pH measurements. Measurements with the surface electrode gave results averaging 0.78 units lower than measurements with the litter suspended in water. The extractable amount of cellulase was significantly less in the samples with a low pH (Fig. 3, Table 1). Approximately 25% of the variation in cellulase was explained by litter pH(S). The pH(H20) data are not used since they are closely correlated with the pH(S) data. Usually fungal cellulases have pH optima at pH 4.5-5.0. Possibly the in situ activity would be even less in the litter samples with a low pH compared to those with a pH approximately 4.5. Previous investigations with acidification of soils (Smith and Whitehead, 1940; White et al., 1949; Schmidt and Ruschmeyer, 1958; Ruschmeyer and Schmidt, 1958) have in some cases shown a decreased cellulolytic activity with decreasing pH. However, some of these experiments on the effect of pH have been carried out using different soils with different pHs. This makes it difficult to come to a conclusion on the effect of pH alone as also other factors varies.
6.0 0 0
t
z .-
z I a
. I
I
I
50
100
150
Moisture Fig. 2. Relationship
between
. T 200
(% of DM)
the pH and the moisture
of the litter.
l pH(S); 0 pH(H20).
Acid rain and litter decomposition
1500
In a comparison of the respiration in the organic layer of different soils, the variation in the respiration could not be explained by the variation in pH (Jorgensen and Wells, 1973). The correlation between moisture and respiration could be significantly improved by using a second degree polynomial. The linear correlation coefficient squared was 0.16 while the coefficient for the polynomial was 0.31 (Table 2). For this reason the moisture squared was included in the multiple regressions, with respiration as the dependent variable. Moisture and pH together explained 37% of the variation in the respiration measurements, and less than 6% was explained by a variation in litter pH(S) between 3.5 and 4.8. The optimum moisture for respiration was approximately 140% of DM (Fig. 4) with a respiration value higher than 90% of maximum between 100 and 180% moisture, when calculated from the fitted equation. However, it does not seem justified to conclude that the respiration was reduced above 140% moisture as there are few measurements. This is in agreement with Heal et al. (1978) who found that the respiration was inhibited by moisture levels below
.
. ‘g 1000 0, ui x s . 8 i
500
&
4.0 Litter pH(S)
3.5
4.5
Fig. 3. Relationship between the cellulase activity and the
pH(S) of the litter.
0
1
25
I
I
I
I
50
100
150
200
Moisture
(% of DM )
Fig. 4. Relationship between the respiration and the moisture of the litter.
Table 2. Correlation coefficient in multiple regressions with cellulase and respiration as dependent variables Dependent variable Cellulase Respiration
Respiration
Step No.
Correlation coefficient, RZ
Variables in regression
1 2
Litter pH(S) Litter pH(S)
Moisture
0.25 0.25
1
Moisture Moisture Moisture
Moisture2 Moisture’
Litter pH(S)
2 3 1 2 3 4
Cellulase Cellulase Cellulase Cellulase
Moisture Moisture Moisture
Moisture’ Moisture’
0.38 0.31 0.16 Litter pH(S)
0.63 0.61 0.46 0.18
J. HOVLAND
26
1000
500 Cellulase
(units
. g DM-’
1500
)
Fig. 5. Relationship between the respiration and the cellulase activity of the litter. lOO%, but appeared to be independent of moisture up to 600%. Spalding (1977, 1978) has shown cellulase activity in coniferous litter to be highly correlated with respiration. The present investigation displays a similar conclusion (Fig. 5, Table 1) when cellulase was included among the variables explaining the variation in respiration, the correlation coefficient was increased considerably (Table 2). My results seem to indicate that under natural conditions the reduced pH in coniferous needle litter due to acid rain may be of small importance for the biological activity measured as cellulase activity and respiration. Acknowledgement-I thank Mr Erik Mmness, The Computing Centre of the Agricultural University, for his assistante with the statistical computations.
REFERENCES ABRAHAMSEN G., BJOR K. and TEIGEN 0. (1976a) Field experiments with simulated acid precipitation in forest ecosystems-1. Soil and vegetation characteristics, experimental design and equipment. SNSF-project FR 4/76, Oslo and As. ABRAHAMSEN G., BJOR K., HORNTVEDT R. and TVEITE B. (1976b) Effects of acid precipitation on coniferous forest. In Impact of Acid Precipitation on Forest and Freshwater Ecosystems in Norway (F. H. Braekke, Ed.), pp. 37-63. SNSF-project FR 6/76, Oslo and As.
ALMIN K. E. and ERIKSSON K.-E. (1967) Enzymic degradation of polymers-I. Viscometric method for the determination of enzymic activity. Biochimica et Biophysicu Actu 139, 238-247. ALMIN K. E.. ERIKSSON K.-E. and JANSSON C. (1967) Enzymic degradation of polymers--II. Viscometric determination of cellulase activity in absolute terms. Biochimicu et Biophysics Acta 139, 248-253. HEAL 0. W., LATTER P. M. and HOW~ON G. (1978) A study of the rates of decomposition of organic matter. In Ecolouical Studies. Vol. 27 (0. W. Heal and D. F. Perkins. E&). pp. 136-161. Springer. Berlin. JORGENSENJ. R. and WELLS C. G. (1973) The relationship of respiration in organic and mineral soil layers to soil chemical properties. PIant & Soil 39, 373-387. MALMER N. (1974) On the effects on water, soil and vegetation of an increasing atmospheric supply of sulphur. A survey on ecological bases. Statens naturvirdssverk SNV PM 402 E, Stockholm. ODEN S. (1968) NederbBrdens och luftens fiirsurning-dess orsaker. fiirlopp och verkan i olika miljiier. Ecoloyical Rrseurch Committee Bulletin. Stockholm 1, l-87. OECD (1977) The OECD programme on long range transport of air pollutants. Measurements and findings. Organisation for economic Co-operation and Development, Paris. RUSCHMEYER 0. R. and SCHMIDT E. L. (1958) Cellulose decomposition in soil burial beds-II. Cellulolytic activity as influenced by alteration of soil properties. Applied Microbiology 6, 115-I 20. SCHMIDT E. L. and RUSCHMEYER 0. R. (1958) Cellulose decomposition in soil burial beds-I. Soil properties in relation to cellulose degradation. Applied Microbiology 6, 108&l 14. SMITH F. B. and WHITEHEAD T. JR (1940) The Effect of substituted cations in the soil complex on the decomposition of organic matter. Proceedings. Soil Science Society of America 5, 248-253. SPALDING B. P. (1977) Enzvmatic activities related to the decomposition of conife;ous leaf litter. Soil Science Society of America Journal 41, 622-627. SPALDING B. P. (1978) The effect of biocidal treatments on respiration and enzymatic activities of Douglas-fir needle litter. Soil Biology d; Biochemistry 10, 537-T43. STUANES A. and SVEISTRUP T. E. (1979) Field experiments with simulated acid rain in forest ecosystems-II. Description and classification of the soils used in field, lysimeter and laboratory experiments. SNSF Project FR 15179, Oslo and As. TAMM C. O., WIKLANDER G. and POPOVIC B. (1977) Effects of application of acid to poor pine forest. Water, Air and Soil Pollution 8, 75-87. TYLER G. (1976) Heavy metal pollution. phosphatase activity, and mineralization of organic phosphorus in forest soils. Soil Biology & Biochemistry 8, 327-332. WHITE J. W.. HOLBECH F. J. and JEFFRIES C. D. (1949) Cellulose-decomposing power in relation to reaction of soils. Soil Science 68, 229-235.