Microb Ecol (1985) 11:259-266
MICROBIAL ECOLOGY 9 1985 Springer-Verlag
Biological Activity in Soil from Forest Stands in Central Sweden, as Related to Site Properties Hans-Orjan Nohrstedt Department of Soil Sciences, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden Abstract. The relationship between biological activities in samples from the forest floor and, respectively, stand (site quality class, tree age, and latitude) and soil (pH, carbon, carbon/nitrogen, and available phosphorus) properties was examined in a multi-site study performed in Central Sweden. The biological activities measured were respiration and phosphatase and dehydrogenase activity. The incubations were made in the laboratory on sieved and homogenized samples. When the biological activities were expressed on a dry matter weight basis, the concentration of organic C was the strongest predictor of respiration and phosphatase activity. In contrast, the strongest predictor of dehydrogenase activity was the pH value. Respiration and dehydrogenase activity were not significantly correlated. When excluding the influence of C concentration by expressing the activities on a C weight basis, respiration and phosphatase activity were not significantly correlated with any of the independent variables studied. Curvilinear models (polynomial equations of second and third order) gave significantly better descriptions of the relationship between respiration and, respectively, pH and C/N ratio, than linear models. Optimum conditions for respiration were indicated at intermediate pH (4-5 in 0.01 M CaC12) and C/N ratio (20-30). The dehydrogenase activity on a C weight basis was correlated with the pH value even more strongly than it was on a dry matter basis. The phosphatase activity was not significantly correlated with the content of available P either on a dry matter basis or on a C weight basis. Introduction
The ongoing acidification of forest ecosystems by industrial pollution is suggested to affect biological processes in soil [21 ]. Results of different studies are, however, largely contradictory [9]. Decomposition of litter is regulated by climate, resource quality, and edaphic factors [20]. There is little information to be found in the literature regarding the influence of acidity on litter decomposition [1, 20, 23]. Studies comparing soil respiration rates at different sites have not shown any significant influence o f pH [10, 22]. Schlesinger has suggested that the difference in morphological appearance of organic matter in mull and in mor soils is due to differences in fragmentation and mixing rather than to differences in the rate of final remineralization [17].
H.-O. Nohrstedt
260 Table 1.
A description of the sampling plots Site quality
Plot no.
Latitude
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
58~ ' 58*35' 58~ ' 58~ 58o21 ' 5904 ' 5904 ' 59"18' 59~ ' 59~ , 59046 ' 59~ ' 60"16' 60~ ' 60016 ' 60~ ' 60*52' 60*29' 60"11' 6004 '
Tree species Picea abies P. abies Pinussilvestris Fagus silvatica P. abies P. abies P. silvestris F r a x i n u s excelsior P. abies + P. silvestris P. abies + P. silvestris P. abies P. silvestris Betula verrucosa P. abies P. abies P. silvestris P. silvestris P. abies + P. silvestris B. verrucosa Quercus robur
Tree age (yr)
class~ (m ~ over bark ha ~ yr -t)
61 55 54 42 40 43 46 ND 96 58 45 45 ND 102 87 77 120 65 65 ND
9.0 9.6 10.1 12.0 12.0 10.1 10.1 10.1 6.5 6.9 9.6 9.6 8.0 6.1 6.1 4.2 4.2 4.4 9.6 11.8
Forest floor (0-3 cm) properties
pH
Organic C (%)
C/N
Available P (~g g-~)
3.76 4.36 4.26 5.63 5.77 3.67 3.66 5.73 3.09 3.20 3.44 3.76 3.89 3.15 3.15 3.05 3.31 3.57 5.50 6.80
15.65 7.70 5.57 5.27 6.37 5.85 4.44 7.52 48.78 44.67 16.32 12.87 14.39 47.58 47.58 49.55 12.11 24.43 9.32 6.11
24.88 16.74 15.05 15.06 15.54 23.28 20.18 11.94 31.07 35.74 25.50 22.19 21.80 34.73 34.73 36.17 37.80 32.57 15.03 13.89
35.1 33.1 25.1 11.0 12.1 40.1 40.0 10.9 85.0 47.2 43.8 41.0 25.0 40.0 40.0 63.8 20.0 28.1 15.0 19.0
N D = not determined a Transformed to the production capacity of Picea abies irrespective of the actual species on the plot
This paper reports on results from an inventory study of soil respiration and phosphatase and dehydrogenase activity as related to site properties of a number of forest stands in Central Sweden. One of the main purposes was to determine whether any obvious relationship was present between biological activity and soil pH in a natural gradient among sites.
Material
and Methods
Forest floor samples were taken from 100 m 2 plots in 20 different stands in Central Sweden. A detailed description of the plots is given in Table 1. The plots were situated from 58*21'-60~ ' N. The stands included both coniferous and deciduous types, and their age was between 40 and 120 years. The site quality class, which is a measure of the stemwood production capacity of a site, varied between 4.2 and 12.0 m 3 over bark ha -t yr -1. The range of some of the chemical properties o f the forest floor was as follows: pH in 0.01 M CaC12, 3.1-6.8; concentration of organic C, 4-50% of dry weight of soil, C / N ratio, 12-38; and content of available P, 11-85 #g g-i soil dry weight. Samples were taken on 3 occasions during the growing season (late May, July, and late September-early October) with a steel cylinder (6 cm diameter) down to a depth o f 3 cm. The samples included the litter-layer. On each sampling occasion, 25 cores were taken from each plot,
Biological Activity in Forest Soil
261
and the cores were then mixed to form a composite sample. The composite sample was passed through a 4 mm sieve and frozen at -20~ After treatment of the samples from the 3 different occasions in this way, they were thawed at +5~ The composite soil samples from the 3 different occasions were then mixed to 1 bulk sample representing the growing season. After storing this bulk sample for 4 weeks at +5~ aliquots were analyzed for respiration and phosphatase and dehydroganase activity. Soil respiration was measured as release of CO2 during a 24-hour incubation at + 12~ About 20 ml loosely stored soil was put into a 115-ml glass bottle equipped with a membrane of butylic rubber. One ml of neon was added as an internal standard. The production of CO2 was determined by taking 0.5 ml gas samples after 3 and 27 hours. The gas samples were immediately injected into a Hewlett-Packard 5880A gas chromatograph equipped with a Thermal Conductivity detector. The column was a Porapak Q (3.2,000 mm, 80/100 mesh). The carrier gas was helium at a flow rate of 30 ml rain -'. The injector, column, and detector temperatures were 130 ~ 60 ~ and 160~ respectively. For measurement of phosphatase activity, 1 g (fresh weight) of soil was put into a 30-ml glass bottle. Eight milliliters of 0.2 M acetate buffer (pH 5.0) and 2 ml 1.44 mM PNP-P (para-nitrophenylphosphate) were added. After 2 hours on a shaker in a water bath at +25~ 2 ml 0.5 M CaC12 and 8 ml 2 M NaOH were added. The solution was passed through a filter and diluted 50 times with 0. t M NaOH. The product of the reaction, PNP (para-nitrophenol), is yellow in basic solution, and the absorbance was measured at 400 nm. Controls were incubated as described, the only difference being that PNP-P was added after the addition of NaOH. Dehydrogenase activity was measured according to Ross [14]. Soil (2.5-4.0 g fresh weight) was put into a 100-mi glass bottle together with 0.75 ml distilled water and 5.0 ml of 0.5% TTC (triphenyltetrazolium chloride) in 0.5 M tris-buffer (tris[hydroxymethyl]aminomethane) with pH 7.6. The bottles were flushed with N2 to obtain an anaerobic incubation. The samples were incubated in the dark at + 15~ for 16 hours. The TPF (triphenylformazan) produced was then extracted by adding 50 ml CH3OH and shaking the bottles for 2 hours. After I hour of sedimentation, 5 ml of the supernatant solution was taken and centrifuged. The absorbance was measured at 485 nm. Controls were treated in the same way, the only exception being that the TTC solution was added after the addition of CH3OH. All measurements of biological activities were made on duplicate aliquots. The differences between these were insignificant when compared to the variation among plots. The site quality class was examined using methods of Hiigglund and Lundmark [6]. The pH value was measured with a glass electrode in the supernatant solution of a 1:3 mixture (by volume) of soil and 0.01 M CaCI2 that had been stirred, equilibrated overnight, and finally stirred once again 1 hour prior to the measurement. Available P was analyzed according to the method of Egn6r et al. [5]. Organic C and N were analyzed with a Carlo Erba Elemental Analyzer Model 1106. The relationships between soil biological activities and stand properties and forest floor chemical properties, respectively, were statistically evaluated by regression analysis [2, 16].
Results T h e d a t a o n b i o l o g i c a l a c t i v i t i e s a r e d e s c r i b e d i n T a b l e 2, b o t h o n a d r y m a t t e r weight basis and on a C weight basis. The variation among plots decreased appreciably for respiration and phosphatase activity when the activities were expressed on C weight basis instead of a dry matter basis. Table 3 gives the linear correlation coefficients for biological activities vs, respectively, stand properties and chemical properties of the forest floor. When expressed on a dry matter weight basis, the respiration and the phosphatase activity were significantly and positively correlated to the concentration of organic C in the forest floor. At the same time, these activities were significantly correlated to variables that follow the C concentration, for example, pH and C/N ratio.
262
H.-O. Nohrstedt
T a b l e 2.
Biological activities in samples from the forest floor (0-3 cm) of 20 stands in Central
Sweden Activity on dry matter weight basis (g-~ h -l)
Parameter Mean value Standard deviation Coefficient of variation (%) Min. value Max. value
Respiration (umol CO2)
Phosphatase (umol PNP)
Activity on C weight basis (g-t C h -I)
Dehydrogenase (nmol TPF)
Respiration 0~mol COz)
Phosphatase (#mol PNP)
Dehydrogenase (nmol TPF)
0.45 0.35
31.2 28.0
14.0 10.0
2.53 0.63
162.9 34.5
172,0 179,0
76.8 0.10 1.22
89.8 7.0 107.6
71.3 0 43.6
25.0 1.28 3.69
21.2 102.3 253.5
104.1 0 713.2
Table 3. Correlation coefficients for the linear relationship between biological activities and, respectively, stand properties and properties of the forest floor Activity on dry matter weight basis Independent variables
Respiration
Phosphatase
Dehydrogenase
Activity on C weight basis Respiration
Phosphatase
Dehydrogenase
Stand properties Quality class Tree age Latitude
-0.801' +0.329 +0.549 ~
-0.804 c +0.561 a +0.559 a
+0.686 c +0.154 -0.192
+0.070 -0.178 -0.051
-0.283 +0,042 +0,326
+0.764 < +0.111 -0.363
+0.928 ~ +0.808" -0.668 b +0_578 ~
+0.906 c +0.752 c -0.598 b +0.504"
-0.643 ~ -0.687 c +0.844 c -0.562 a
-0.428 -0.018 -0.134 -0.279
-0.140 +0.070 -0.025 -0.418
-0.663 b -0.741 c +0.887 c -0.585 ~
Forest floor C C/N pH Available P P < 0.05 b p < 0.01 ~P < 0.001
When examining the influence of properties other than the C concentration, it is a better approach to exclude the overwhelming influence of C by expressing the activities on a C weight basis. In doing so there is obviously no influence (Table 3) o f the stand and soil properties on respiration and phosphatase activity, as indicated by linear regression analysis. Dehydrogenase activity was quite different from the 2 other activities. Both on a dry matter weight basis and on a C weight basis (Fig. 1), the dehydrogenase activity was strongly and positively correlated with pH. A multiple regression analysis showed that when the dehydrogenase activity was expressed on a dry matter weight basis, the only significant predictor was pH. On a C weight basis, both available P and the C / N ratio made a significant contribution in addition to pH, although the influence of pH was still dominant. From the above dis-
Biological Activity in Forest Soil
rL_J
700
600
"
263
l'-ffl
y=142.2.X-417.3 r= 0.8857
.
/
s00 o c
>_
z~O0
{--
u
300
t/'l
< 200 m >- 100 -IN
Fig. I. R e l a t i o n s h i p between dehydrogenase activity on a carb o n weight basis and pH.
0 pH (001 H CaCt2)
'r 9-
i--
800
"6 E
o 600 >-
/+00
~< 200
9
J.
8
9 9
o
9
, t . . . . . , I 2 3 4 RESPIRATION (pmoles [0 2 g-1 Eh-l)
Fig. 2. R e l a t i o n s h i p between dchydrogcnasc activity on a carb o n weight basis and respiration.
cussion, it is clear that dehydrogenase activity and respiration are not correlated to each other (Fig. 2). When plotting the biological activities vs stand and forest floor properties, the only other types of relationships indicated were between respiration on C basis and pH or C/N ratio. As shown in Fig. 3, curvilinear relationships are indicated by the plotted data. Polynomial equations including terms of both second and third order were fitted to the data, and the models given in Fig. 3 gave a significantly (P < 0.05) higher degree of explanation than the first order
264
H.-(). Nohrstedt
*on
~o r
i
3
o E
i
i
|
/., 5 pH (0.01H CaCI2)
i
6
7
Gt.
_1 =~176
.
i
i
i
10
20
30
9
i
t+O
i
F i g . 3. R e l a t i o n s h i p b e t w e e n respiration on a carbon weight basis and, respectively, pH and C / N r a t i o . T h e e q u a t i o n s for t h e fitted l i n e s w e r e as follows: r e s p i r a t i o n v s p H : Y -- - 3 0 . 8 + 21.2-X - 4.29.X 2 + 0.28-X 3 w i t h R~ 2 = 37.5%; r e s p i r a t i o n v s C/N ratio: 1/Y = -0.96 + 0.32. X - 0 . 6 4 . 1 0 - 2 . X 2 w i t h R= 2 = 17.9%, a n d 2 / Y = - l 1.55 + 1 . 7 9 - X - 7 . 0 - 1 0 - 2 . X 2 -]- 8.6. 1 0 - 4 - X 3 w i t h R , 2 = 39.4%.
50
C/N
equations. In the case of respiration vs pH, introducing a second order term did not give a significantly better explanation, but including both a second and third order term gave a significant contribution (P < 0.01). In the case of respiration vs C/N ratio, both the second and the third order equations were significantly better models than the first order equation, and the third order equation was significantly better than the second order (P < 0.05). According to these models the respiration was highest at intermediate pH (4-5) and at intermediate C/N ratio (20-30). At pH values below about 4, the respiration clearly decreased with increasing acidity. For pH values below 4.2 there was a significant positive linear correlation between respiration and pH (r = 0.666, P < 0.01). However, a decrease o f p H at the same time corresponded with an increase in the C/N ratio. Therefore, a multiple regression analysis was made with respiration as the dependent variable and pH and C/N ratio as independent variables for data with pH values lower than 4.2 and C / N ratios above 20. The analysis showed that pH had a significant influence (P < 0.05) and that the C/ N ratio was without influence.
Discussion
When excluding the influence of climate by incubating the soil samples in the laboratory and taking into account the differences in C concentration in soil by expressing the biological activities on a C weight basis, there were no obvious differences in mineralization rates of C (measured as CO2) or P (phosphatase
BiologicalActivityin Forest Soil
265
activity) as related to the chemical properties of soil (pH, C, C/N, and available P) and the stand properties (site quality class, tree age, and latitude). The biological potential for final decomposition of organic materials seems to be similar over a range of conditions, which is consistent with the view held by Schlesinger [17], mentioned in the introduction. However, curvilinear models (polynomial equations of second and third order) gave statistically significantly better descriptions o f the relationship between respiration and C / N ratio or pH than was the case for linear models. Optimum conditions for respiration were indicated in the pH range 4-5 and the C/N ratio range 20-30. This C/N ratio range includes the value of 25 given by Swift et al. [20] as the most suitable for microbial growth. In fact, the second order equation had its optimum at a C/N ratio of 24.96. The negative relationship between respiration and acidity at pH values below 4.2 suggests that an acidification of soils with intermediate pH values may result in suppressed mineralization rates of organic material. The positive correlation between phosphatase activity on a dry matter basis and concentration of C in soil is consistent with other reports [3, 7, 11] and may be due to both a higher microbial activity, and thereby a higher production of phosphatases, and to the capability of organic matter to adsorb extracellular phosphatases in an active form. Phosphatase activity is considered to be inversely related to the supply of P in available forms [18]. Phosphate ions inhibit the synthesis of phosphatases in soil [ 19]. The results presented in this paper indicate a negative relationship between phosphatase activity on a C weight basis and available P, but the correlation was not significant (P > 0.05). Dehydrogenase activity in soil is defined as the reduction of an artificial electron acceptor under controlled conditions [13]. Although dehydrogenase activity is considered as a measure of biological activity [ 12], its physiological and ecological interpretation seems unclear since no consistent relationships have been found to exist with respiration or number of microbes [8, 18]. In individual soils, the dehydrogenase activity has been reported to decrease with depth, thereby being related to the concentration of organic matter [3, 15]. However, when comparing the dehydrogenase activity of different soils, acidity seems to have been the primary regulating factor [3, 8, 13, 15]. The dehydrogenase activity was significantly lower in podzolic soils and gley soils than in brown forest soils and rendzina soils [3, 4]. The close relationship that was found in the present study between dehydrogenase activity and, respectively, pH and site productivity is consistent with the literature references cited and may suggest the use of dehydrogenase activity as a biological index for soil fertility. The total lack of relation between dehydrogenase activity and respiration has to be considered as a challenge for further research. There are still no explanations of this anomaly. It is possible that the artificial electron acceptor (TTC) had different access to soil bacteria and soil fungi or that the biological availability of TTC and/or the analytical recovery of the reaction product (TPF) was strongly influenced by differences in soil properties. Acknowledgments. Financialsupport for the workpresentedin this paperwas givenby the National Swedish EnvironmentProtection Board.
266
H.-O. Nohrstedt
References 1. Dickinson CH (1974) Decomposition of litter in soil. In: Dickinson CH and Pugh GJF (eds) Biology of plant litter decomposition. Vol. 2. Academic Press, London and New York, pp 633-658 2. Dunn OJ and Clark VA (1974) Applied statistics: analysis of variance and regression. John Wiley, New York 3. Dutzler-Frartz G (1977) Der Einfluss einiger chemischer und physikalischer Bodenmerkmale auf die Enzymaktivitlit verschiedener Bodentypen. Z Pflanzenernaer Bodenkd 140:329-350 4. Dutzler-Franz G (1977) Beziehungen zwischen der Enzymaktivi~t verschiedener Bodentypen, der Mikrobiellen Aktivit~t, der Murzelmasse und einigen Klimafaktoren. Z Pflanzenernaer Bodenkd 140:351-374 5. Egn6r H, Riehm H, Domingo WR (1960) Untersuchungen fiber die chemische Bodenanalyse als Grundlage for die Beurteilung des N~hrstoffzustandes der B6den. II. Chemische Extraktionsmetodon zur Phosphor- und Kaliumbestimmung. Annals Roy Agric Coll Sweden 26: 199-215 6. Hltgglund B, Lundmark J-E (1981) Handledning i bonitering reed Skogshggskolans boniteringssystem. Skogsstyrelsen, J~Snkgping (in Swedish). 7. Harrison AF (1983) Relationship between intensity of phosphatase activity and physicochemical properties in woodland soils. Soil Biol Biochem 15:93-99 8. Howard PJA (1972) Problems in the estimation of biological activity in soil. Oikos 23:235240 9. Hutchinson TC, Havas M (eds) (1980) Effects of acid precipitation on terrestrial ecosystems. Proceedings of the N A T O conference on effects of acid precipitation on vegetation and soils, Toronto, Ontario, Canada, May 21-27, 1978, Plenum Press, New York. 10. Jorgensen JR, Wells CG (1973) The relationship of respiration in organic and mineral soil layers to soil chemical properties. Plant Soil 39:373-387 11. Juma NG, Tabatabai MA (1978) Distribution of phosphomonoesterases in soil. Soil Sci 126: 101-108 12. Ladd JN (1978) Origin and range ofenzymes in soil. In: Bums RG (ed) Soil enzymes. Academic Press, London, pp 51-96 13. Moore AW, Russell JS (1972) Factors affecting dehydrogenase activity as an index of soil fertility. Plant Soil 37:675-682 14. Ross DJ (1970) Effects of storage on dehydrogenase activities of soils. Soil Biol Biochem 2: 55-61 15. Ross DJ (1973) Biochemical activities in a soil profile under hard beech forest. 2. Some factors influencing oxygen uptakes and dehydrogenase activities. N Z J Sci 17:225-240 16. Ryan Jr TA, Joiner BL, Ryan BF (1981) Minitab reference manual. The Pennsylvania State University. 17. Schlesinger WH (1977) Carbon balance in terrestrial detritus. Ann Rev Ecol Syst 8:51-81 18. Skujins J (1967) Enzymes in soil. In: McLaren AD, Peterson GH (eds) Soil biochemistry. Marcel Dekker, New York, pp 371-414 19. Spiers GA, McGill WB (1979) Effects of phosphorus addition and energy supply on acid phosphatase production and activity in soils. Soil Biol Biochem 11:3-8 20. Swift MJ, Heal OW, Anderson JM (1979) Decomposition in terrestrial ecosystems. Studies in ecology. Vol. 5. Blackwell Scientific Publications, Oxford 21. Tamm CO (1976) Acid precipitation: biological effects in soil and on forest vegetation. Ambio 5:235-238 22. Van Cleve K, Sprague D (1971) Respiration rates in the forest floor of birch and aspen stands in interior Alaska. Arct Alp Res 3:17-26 23. Williams ST, Gray TRG (1974) Decomposition of litter on the soil surface. In: Dickinson CH, Pugh GJF (eds) Biology of plant litter decomposition. Vol. 2. Academic Press, London and New York, pp 611-632
MICROBIAL ECOLOGY 9 1985 Springer-Verlag
Biological Activity in Soil from Forest Stands in Central Sweden, as Related to Site Properties Hans-Orjan Nohrstedt Department of Soil Sciences, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden Abstract. The relationship between biological activities in samples from the forest floor and, respectively, stand (site quality class, tree age, and latitude) and soil (pH, carbon, carbon/nitrogen, and available phosphorus) properties was examined in a multi-site study performed in Central Sweden. The biological activities measured were respiration and phosphatase and dehydrogenase activity. The incubations were made in the laboratory on sieved and homogenized samples. When the biological activities were expressed on a dry matter weight basis, the concentration of organic C was the strongest predictor of respiration and phosphatase activity. In contrast, the strongest predictor of dehydrogenase activity was the pH value. Respiration and dehydrogenase activity were not significantly correlated. When excluding the influence of C concentration by expressing the activities on a C weight basis, respiration and phosphatase activity were not significantly correlated with any of the independent variables studied. Curvilinear models (polynomial equations of second and third order) gave significantly better descriptions of the relationship between respiration and, respectively, pH and C/N ratio, than linear models. Optimum conditions for respiration were indicated at intermediate pH (4-5 in 0.01 M CaC12) and C/N ratio (20-30). The dehydrogenase activity on a C weight basis was correlated with the pH value even more strongly than it was on a dry matter basis. The phosphatase activity was not significantly correlated with the content of available P either on a dry matter basis or on a C weight basis. Introduction
The ongoing acidification of forest ecosystems by industrial pollution is suggested to affect biological processes in soil [21 ]. Results of different studies are, however, largely contradictory [9]. Decomposition of litter is regulated by climate, resource quality, and edaphic factors [20]. There is little information to be found in the literature regarding the influence of acidity on litter decomposition [1, 20, 23]. Studies comparing soil respiration rates at different sites have not shown any significant influence o f pH [10, 22]. Schlesinger has suggested that the difference in morphological appearance of organic matter in mull and in mor soils is due to differences in fragmentation and mixing rather than to differences in the rate of final remineralization [17].
H.-O. Nohrstedt
260 Table 1.
A description of the sampling plots Site quality
Plot no.
Latitude
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
58~ ' 58*35' 58~ ' 58~ 58o21 ' 5904 ' 5904 ' 59"18' 59~ ' 59~ , 59046 ' 59~ ' 60"16' 60~ ' 60016 ' 60~ ' 60*52' 60*29' 60"11' 6004 '
Tree species Picea abies P. abies Pinussilvestris Fagus silvatica P. abies P. abies P. silvestris F r a x i n u s excelsior P. abies + P. silvestris P. abies + P. silvestris P. abies P. silvestris Betula verrucosa P. abies P. abies P. silvestris P. silvestris P. abies + P. silvestris B. verrucosa Quercus robur
Tree age (yr)
class~ (m ~ over bark ha ~ yr -t)
61 55 54 42 40 43 46 ND 96 58 45 45 ND 102 87 77 120 65 65 ND
9.0 9.6 10.1 12.0 12.0 10.1 10.1 10.1 6.5 6.9 9.6 9.6 8.0 6.1 6.1 4.2 4.2 4.4 9.6 11.8
Forest floor (0-3 cm) properties
pH
Organic C (%)
C/N
Available P (~g g-~)
3.76 4.36 4.26 5.63 5.77 3.67 3.66 5.73 3.09 3.20 3.44 3.76 3.89 3.15 3.15 3.05 3.31 3.57 5.50 6.80
15.65 7.70 5.57 5.27 6.37 5.85 4.44 7.52 48.78 44.67 16.32 12.87 14.39 47.58 47.58 49.55 12.11 24.43 9.32 6.11
24.88 16.74 15.05 15.06 15.54 23.28 20.18 11.94 31.07 35.74 25.50 22.19 21.80 34.73 34.73 36.17 37.80 32.57 15.03 13.89
35.1 33.1 25.1 11.0 12.1 40.1 40.0 10.9 85.0 47.2 43.8 41.0 25.0 40.0 40.0 63.8 20.0 28.1 15.0 19.0
N D = not determined a Transformed to the production capacity of Picea abies irrespective of the actual species on the plot
This paper reports on results from an inventory study of soil respiration and phosphatase and dehydrogenase activity as related to site properties of a number of forest stands in Central Sweden. One of the main purposes was to determine whether any obvious relationship was present between biological activity and soil pH in a natural gradient among sites.
Material
and Methods
Forest floor samples were taken from 100 m 2 plots in 20 different stands in Central Sweden. A detailed description of the plots is given in Table 1. The plots were situated from 58*21'-60~ ' N. The stands included both coniferous and deciduous types, and their age was between 40 and 120 years. The site quality class, which is a measure of the stemwood production capacity of a site, varied between 4.2 and 12.0 m 3 over bark ha -t yr -1. The range of some of the chemical properties o f the forest floor was as follows: pH in 0.01 M CaC12, 3.1-6.8; concentration of organic C, 4-50% of dry weight of soil, C / N ratio, 12-38; and content of available P, 11-85 #g g-i soil dry weight. Samples were taken on 3 occasions during the growing season (late May, July, and late September-early October) with a steel cylinder (6 cm diameter) down to a depth o f 3 cm. The samples included the litter-layer. On each sampling occasion, 25 cores were taken from each plot,
Biological Activity in Forest Soil
261
and the cores were then mixed to form a composite sample. The composite sample was passed through a 4 mm sieve and frozen at -20~ After treatment of the samples from the 3 different occasions in this way, they were thawed at +5~ The composite soil samples from the 3 different occasions were then mixed to 1 bulk sample representing the growing season. After storing this bulk sample for 4 weeks at +5~ aliquots were analyzed for respiration and phosphatase and dehydroganase activity. Soil respiration was measured as release of CO2 during a 24-hour incubation at + 12~ About 20 ml loosely stored soil was put into a 115-ml glass bottle equipped with a membrane of butylic rubber. One ml of neon was added as an internal standard. The production of CO2 was determined by taking 0.5 ml gas samples after 3 and 27 hours. The gas samples were immediately injected into a Hewlett-Packard 5880A gas chromatograph equipped with a Thermal Conductivity detector. The column was a Porapak Q (3.2,000 mm, 80/100 mesh). The carrier gas was helium at a flow rate of 30 ml rain -'. The injector, column, and detector temperatures were 130 ~ 60 ~ and 160~ respectively. For measurement of phosphatase activity, 1 g (fresh weight) of soil was put into a 30-ml glass bottle. Eight milliliters of 0.2 M acetate buffer (pH 5.0) and 2 ml 1.44 mM PNP-P (para-nitrophenylphosphate) were added. After 2 hours on a shaker in a water bath at +25~ 2 ml 0.5 M CaC12 and 8 ml 2 M NaOH were added. The solution was passed through a filter and diluted 50 times with 0. t M NaOH. The product of the reaction, PNP (para-nitrophenol), is yellow in basic solution, and the absorbance was measured at 400 nm. Controls were incubated as described, the only difference being that PNP-P was added after the addition of NaOH. Dehydrogenase activity was measured according to Ross [14]. Soil (2.5-4.0 g fresh weight) was put into a 100-mi glass bottle together with 0.75 ml distilled water and 5.0 ml of 0.5% TTC (triphenyltetrazolium chloride) in 0.5 M tris-buffer (tris[hydroxymethyl]aminomethane) with pH 7.6. The bottles were flushed with N2 to obtain an anaerobic incubation. The samples were incubated in the dark at + 15~ for 16 hours. The TPF (triphenylformazan) produced was then extracted by adding 50 ml CH3OH and shaking the bottles for 2 hours. After I hour of sedimentation, 5 ml of the supernatant solution was taken and centrifuged. The absorbance was measured at 485 nm. Controls were treated in the same way, the only exception being that the TTC solution was added after the addition of CH3OH. All measurements of biological activities were made on duplicate aliquots. The differences between these were insignificant when compared to the variation among plots. The site quality class was examined using methods of Hiigglund and Lundmark [6]. The pH value was measured with a glass electrode in the supernatant solution of a 1:3 mixture (by volume) of soil and 0.01 M CaCI2 that had been stirred, equilibrated overnight, and finally stirred once again 1 hour prior to the measurement. Available P was analyzed according to the method of Egn6r et al. [5]. Organic C and N were analyzed with a Carlo Erba Elemental Analyzer Model 1106. The relationships between soil biological activities and stand properties and forest floor chemical properties, respectively, were statistically evaluated by regression analysis [2, 16].
Results T h e d a t a o n b i o l o g i c a l a c t i v i t i e s a r e d e s c r i b e d i n T a b l e 2, b o t h o n a d r y m a t t e r weight basis and on a C weight basis. The variation among plots decreased appreciably for respiration and phosphatase activity when the activities were expressed on C weight basis instead of a dry matter basis. Table 3 gives the linear correlation coefficients for biological activities vs, respectively, stand properties and chemical properties of the forest floor. When expressed on a dry matter weight basis, the respiration and the phosphatase activity were significantly and positively correlated to the concentration of organic C in the forest floor. At the same time, these activities were significantly correlated to variables that follow the C concentration, for example, pH and C/N ratio.
262
H.-O. Nohrstedt
T a b l e 2.
Biological activities in samples from the forest floor (0-3 cm) of 20 stands in Central
Sweden Activity on dry matter weight basis (g-~ h -l)
Parameter Mean value Standard deviation Coefficient of variation (%) Min. value Max. value
Respiration (umol CO2)
Phosphatase (umol PNP)
Activity on C weight basis (g-t C h -I)
Dehydrogenase (nmol TPF)
Respiration 0~mol COz)
Phosphatase (#mol PNP)
Dehydrogenase (nmol TPF)
0.45 0.35
31.2 28.0
14.0 10.0
2.53 0.63
162.9 34.5
172,0 179,0
76.8 0.10 1.22
89.8 7.0 107.6
71.3 0 43.6
25.0 1.28 3.69
21.2 102.3 253.5
104.1 0 713.2
Table 3. Correlation coefficients for the linear relationship between biological activities and, respectively, stand properties and properties of the forest floor Activity on dry matter weight basis Independent variables
Respiration
Phosphatase
Dehydrogenase
Activity on C weight basis Respiration
Phosphatase
Dehydrogenase
Stand properties Quality class Tree age Latitude
-0.801' +0.329 +0.549 ~
-0.804 c +0.561 a +0.559 a
+0.686 c +0.154 -0.192
+0.070 -0.178 -0.051
-0.283 +0,042 +0,326
+0.764 < +0.111 -0.363
+0.928 ~ +0.808" -0.668 b +0_578 ~
+0.906 c +0.752 c -0.598 b +0.504"
-0.643 ~ -0.687 c +0.844 c -0.562 a
-0.428 -0.018 -0.134 -0.279
-0.140 +0.070 -0.025 -0.418
-0.663 b -0.741 c +0.887 c -0.585 ~
Forest floor C C/N pH Available P P < 0.05 b p < 0.01 ~P < 0.001
When examining the influence of properties other than the C concentration, it is a better approach to exclude the overwhelming influence of C by expressing the activities on a C weight basis. In doing so there is obviously no influence (Table 3) o f the stand and soil properties on respiration and phosphatase activity, as indicated by linear regression analysis. Dehydrogenase activity was quite different from the 2 other activities. Both on a dry matter weight basis and on a C weight basis (Fig. 1), the dehydrogenase activity was strongly and positively correlated with pH. A multiple regression analysis showed that when the dehydrogenase activity was expressed on a dry matter weight basis, the only significant predictor was pH. On a C weight basis, both available P and the C / N ratio made a significant contribution in addition to pH, although the influence of pH was still dominant. From the above dis-
Biological Activity in Forest Soil
rL_J
700
600
"
263
l'-ffl
y=142.2.X-417.3 r= 0.8857
.
/
s00 o c
>_
z~O0
{--
u
300
t/'l
< 200 m >- 100 -IN
Fig. I. R e l a t i o n s h i p between dehydrogenase activity on a carb o n weight basis and pH.
0 pH (001 H CaCt2)
'r 9-
i--
800
"6 E
o 600 >-
/+00
~< 200
9
J.
8
9 9
o
9
, t . . . . . , I 2 3 4 RESPIRATION (pmoles [0 2 g-1 Eh-l)
Fig. 2. R e l a t i o n s h i p between dchydrogcnasc activity on a carb o n weight basis and respiration.
cussion, it is clear that dehydrogenase activity and respiration are not correlated to each other (Fig. 2). When plotting the biological activities vs stand and forest floor properties, the only other types of relationships indicated were between respiration on C basis and pH or C/N ratio. As shown in Fig. 3, curvilinear relationships are indicated by the plotted data. Polynomial equations including terms of both second and third order were fitted to the data, and the models given in Fig. 3 gave a significantly (P < 0.05) higher degree of explanation than the first order
264
H.-(). Nohrstedt
*on
~o r
i
3
o E
i
i
|
/., 5 pH (0.01H CaCI2)
i
6
7
Gt.
_1 =~176
.
i
i
i
10
20
30
9
i
t+O
i
F i g . 3. R e l a t i o n s h i p b e t w e e n respiration on a carbon weight basis and, respectively, pH and C / N r a t i o . T h e e q u a t i o n s for t h e fitted l i n e s w e r e as follows: r e s p i r a t i o n v s p H : Y -- - 3 0 . 8 + 21.2-X - 4.29.X 2 + 0.28-X 3 w i t h R~ 2 = 37.5%; r e s p i r a t i o n v s C/N ratio: 1/Y = -0.96 + 0.32. X - 0 . 6 4 . 1 0 - 2 . X 2 w i t h R= 2 = 17.9%, a n d 2 / Y = - l 1.55 + 1 . 7 9 - X - 7 . 0 - 1 0 - 2 . X 2 -]- 8.6. 1 0 - 4 - X 3 w i t h R , 2 = 39.4%.
50
C/N
equations. In the case of respiration vs pH, introducing a second order term did not give a significantly better explanation, but including both a second and third order term gave a significant contribution (P < 0.01). In the case of respiration vs C/N ratio, both the second and the third order equations were significantly better models than the first order equation, and the third order equation was significantly better than the second order (P < 0.05). According to these models the respiration was highest at intermediate pH (4-5) and at intermediate C/N ratio (20-30). At pH values below about 4, the respiration clearly decreased with increasing acidity. For pH values below 4.2 there was a significant positive linear correlation between respiration and pH (r = 0.666, P < 0.01). However, a decrease o f p H at the same time corresponded with an increase in the C/N ratio. Therefore, a multiple regression analysis was made with respiration as the dependent variable and pH and C/N ratio as independent variables for data with pH values lower than 4.2 and C / N ratios above 20. The analysis showed that pH had a significant influence (P < 0.05) and that the C/ N ratio was without influence.
Discussion
When excluding the influence of climate by incubating the soil samples in the laboratory and taking into account the differences in C concentration in soil by expressing the biological activities on a C weight basis, there were no obvious differences in mineralization rates of C (measured as CO2) or P (phosphatase
BiologicalActivityin Forest Soil
265
activity) as related to the chemical properties of soil (pH, C, C/N, and available P) and the stand properties (site quality class, tree age, and latitude). The biological potential for final decomposition of organic materials seems to be similar over a range of conditions, which is consistent with the view held by Schlesinger [17], mentioned in the introduction. However, curvilinear models (polynomial equations of second and third order) gave statistically significantly better descriptions o f the relationship between respiration and C / N ratio or pH than was the case for linear models. Optimum conditions for respiration were indicated in the pH range 4-5 and the C/N ratio range 20-30. This C/N ratio range includes the value of 25 given by Swift et al. [20] as the most suitable for microbial growth. In fact, the second order equation had its optimum at a C/N ratio of 24.96. The negative relationship between respiration and acidity at pH values below 4.2 suggests that an acidification of soils with intermediate pH values may result in suppressed mineralization rates of organic material. The positive correlation between phosphatase activity on a dry matter basis and concentration of C in soil is consistent with other reports [3, 7, 11] and may be due to both a higher microbial activity, and thereby a higher production of phosphatases, and to the capability of organic matter to adsorb extracellular phosphatases in an active form. Phosphatase activity is considered to be inversely related to the supply of P in available forms [18]. Phosphate ions inhibit the synthesis of phosphatases in soil [ 19]. The results presented in this paper indicate a negative relationship between phosphatase activity on a C weight basis and available P, but the correlation was not significant (P > 0.05). Dehydrogenase activity in soil is defined as the reduction of an artificial electron acceptor under controlled conditions [13]. Although dehydrogenase activity is considered as a measure of biological activity [ 12], its physiological and ecological interpretation seems unclear since no consistent relationships have been found to exist with respiration or number of microbes [8, 18]. In individual soils, the dehydrogenase activity has been reported to decrease with depth, thereby being related to the concentration of organic matter [3, 15]. However, when comparing the dehydrogenase activity of different soils, acidity seems to have been the primary regulating factor [3, 8, 13, 15]. The dehydrogenase activity was significantly lower in podzolic soils and gley soils than in brown forest soils and rendzina soils [3, 4]. The close relationship that was found in the present study between dehydrogenase activity and, respectively, pH and site productivity is consistent with the literature references cited and may suggest the use of dehydrogenase activity as a biological index for soil fertility. The total lack of relation between dehydrogenase activity and respiration has to be considered as a challenge for further research. There are still no explanations of this anomaly. It is possible that the artificial electron acceptor (TTC) had different access to soil bacteria and soil fungi or that the biological availability of TTC and/or the analytical recovery of the reaction product (TPF) was strongly influenced by differences in soil properties. Acknowledgments. Financialsupport for the workpresentedin this paperwas givenby the National Swedish EnvironmentProtection Board.
266
H.-O. Nohrstedt
References 1. Dickinson CH (1974) Decomposition of litter in soil. In: Dickinson CH and Pugh GJF (eds) Biology of plant litter decomposition. Vol. 2. Academic Press, London and New York, pp 633-658 2. Dunn OJ and Clark VA (1974) Applied statistics: analysis of variance and regression. John Wiley, New York 3. Dutzler-Frartz G (1977) Der Einfluss einiger chemischer und physikalischer Bodenmerkmale auf die Enzymaktivitlit verschiedener Bodentypen. Z Pflanzenernaer Bodenkd 140:329-350 4. Dutzler-Franz G (1977) Beziehungen zwischen der Enzymaktivi~t verschiedener Bodentypen, der Mikrobiellen Aktivit~t, der Murzelmasse und einigen Klimafaktoren. Z Pflanzenernaer Bodenkd 140:351-374 5. Egn6r H, Riehm H, Domingo WR (1960) Untersuchungen fiber die chemische Bodenanalyse als Grundlage for die Beurteilung des N~hrstoffzustandes der B6den. II. Chemische Extraktionsmetodon zur Phosphor- und Kaliumbestimmung. Annals Roy Agric Coll Sweden 26: 199-215 6. Hltgglund B, Lundmark J-E (1981) Handledning i bonitering reed Skogshggskolans boniteringssystem. Skogsstyrelsen, J~Snkgping (in Swedish). 7. Harrison AF (1983) Relationship between intensity of phosphatase activity and physicochemical properties in woodland soils. Soil Biol Biochem 15:93-99 8. Howard PJA (1972) Problems in the estimation of biological activity in soil. Oikos 23:235240 9. Hutchinson TC, Havas M (eds) (1980) Effects of acid precipitation on terrestrial ecosystems. Proceedings of the N A T O conference on effects of acid precipitation on vegetation and soils, Toronto, Ontario, Canada, May 21-27, 1978, Plenum Press, New York. 10. Jorgensen JR, Wells CG (1973) The relationship of respiration in organic and mineral soil layers to soil chemical properties. Plant Soil 39:373-387 11. Juma NG, Tabatabai MA (1978) Distribution of phosphomonoesterases in soil. Soil Sci 126: 101-108 12. Ladd JN (1978) Origin and range ofenzymes in soil. In: Bums RG (ed) Soil enzymes. Academic Press, London, pp 51-96 13. Moore AW, Russell JS (1972) Factors affecting dehydrogenase activity as an index of soil fertility. Plant Soil 37:675-682 14. Ross DJ (1970) Effects of storage on dehydrogenase activities of soils. Soil Biol Biochem 2: 55-61 15. Ross DJ (1973) Biochemical activities in a soil profile under hard beech forest. 2. Some factors influencing oxygen uptakes and dehydrogenase activities. N Z J Sci 17:225-240 16. Ryan Jr TA, Joiner BL, Ryan BF (1981) Minitab reference manual. The Pennsylvania State University. 17. Schlesinger WH (1977) Carbon balance in terrestrial detritus. Ann Rev Ecol Syst 8:51-81 18. Skujins J (1967) Enzymes in soil. In: McLaren AD, Peterson GH (eds) Soil biochemistry. Marcel Dekker, New York, pp 371-414 19. Spiers GA, McGill WB (1979) Effects of phosphorus addition and energy supply on acid phosphatase production and activity in soils. Soil Biol Biochem 11:3-8 20. Swift MJ, Heal OW, Anderson JM (1979) Decomposition in terrestrial ecosystems. Studies in ecology. Vol. 5. Blackwell Scientific Publications, Oxford 21. Tamm CO (1976) Acid precipitation: biological effects in soil and on forest vegetation. Ambio 5:235-238 22. Van Cleve K, Sprague D (1971) Respiration rates in the forest floor of birch and aspen stands in interior Alaska. Arct Alp Res 3:17-26 23. Williams ST, Gray TRG (1974) Decomposition of litter on the soil surface. In: Dickinson CH, Pugh GJF (eds) Biology of plant litter decomposition. Vol. 2. Academic Press, London and New York, pp 611-632
