Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue6.Jun2000:

Soil Biology & Biochemistry 32 (2000) 733±745
www.elsevier.com/locate/soilbio

Biochemical properties of acid soils under climax vegetation
(Atlantic oakwood) in an area of the European temperate±humid
zone (Galicia, NW Spain): general parameters
M.C. LeiroÂs a,*, C. Trasar-Cepeda b, S. Seoane a, F. Gil-Sotres a
a

Departamento de EdafologõÂa y QuõÂmica AgrõÂcola, Universidad de Santiago de Compostela, E-15706 Santiago de Compostela, Spain
Departamento de BioquõÂmica del Suelo, Instituto de Investigaciones AgrobioloÂgicas de Galicia, Consejo Superior de Investigaciones Cientõ®cas,
Apartado 122, E-15080 Santiago de Compostela, Spain

b

Accepted 6 October 1999

Abstract
The concept of sustainable development suggests that soil quality should be measured on the basis of the most
environmentally sensitive properties of native soils under climax vegetation. The most relevant properties are biochemical. We
describe the general biochemical parameters of the O and Ah horizons of 40 native Umbrisols under climax Atlantic oakwood

in Galicia (NW Spain). The properties studied were: microbial biomass C (O horizons 19352450 mg C kgÿ1, Ah horizons 7812
253 mg C kgÿ1), N ¯ush (68220 and 26213 mg kgÿ1), soil respiration (12.924.5 and 2.620.8 mg CO2-C gÿ1 hÿ1), ATP (8.91
23.20 and 2.7721.38 mg gÿ1), dehydrogenase activity (5552205 and 207258 nmol INTF gÿ1 hÿ1), catalase activity (3.921.1
and 2.020.9 mmol H2O2 consumed gÿ1 hÿ1), N mineralization capacity (113266 and 30213 mg N kgÿ1 10 dÿ1), and arginine
ÿ1 ÿ1
ammoni®cation rate (11.125.9 and 4.922.2 mg N-NH+
h ). The values reported are generally within the ranges found in
4 g
the literature. The correlations between biochemical parameters and chemical variables show in these soils microbial population
size and activity directly related to both organic matter and available nutrient contents. 7 2000 Elsevier Science Ltd. All rights
reserved.
Keywords: Soil biochemical properties; Soil microbial activity; Soil nitrogen mineralization; Temperate forest soils

1. Introduction
There is intense interest in the identi®cation of soil
quality markers and the de®nition of soil quality indices. As regards the latter, many approaches seek more
or less complex combinations of physical, chemical
and biochemical properties that jointly evaluate the
three basic functions de®ning sustainable soil quality
(Pankhurst et al., 1997), these qualities are production

(the capacity to yield healthy, abundant crops), ®ltration (the capacity of the soil to remove any pollu-

* Corresponding author. Tel.: +34-981-963-100, ext. 15042; fax:
+34-981-594-912.
E-mail address: edleiros@usc.es (M.C. LeiroÂs).

tant from waters that pass through it) and degradation
(the capacity of the soil to function properly as part of
a mature, self-sustaining ecosystem). It is recognized,
however, that a simpler approach is to use only biochemical properties, which are the most sensitive to environmental stress (Vanhala and Ahtiainen, 1994) and
play the greatest role in degradation (Yakovchenko et
al., 1996).
According to Visser and Parkinson (1992), the
biochemical properties of the soil can be studied at
three di€erent levels: microbial populations, biotic
communities, and the properties involved in organic
matter and nutrient cycles. In spite of the ecological
interest of the ®rst two levels, their immediate relevance to soil quality evaluation is doubtful; much
more relevant is the characterization of the soil

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 9 5 - 9

734

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

properties involved in the transformation of organic
matter. The biochemical properties corresponding to
this third level can be divided in two groups (Nannipieri et al., 1995): general parameters, which
include all the variables directly related to microbial
activity (microbial biomass C and N, respiration,
nitrogen mineralization capacity, etc.), and speci®c
parameters, which include the activities of extracellular hydrolytic enzymes that are involved in the
carbon, nitrogen, sulphur and phosphorus cycles
and are to some extent independent of microbial
population dynamics because of their stabilization
by soil colloids (Burns, 1982).
The estimation of soil quality on the basis of biochemical properties alone is currently limited by the
lack of studies considering the simultaneous variation

of a wide range of biochemical properties. Furthermore, comparisons among the results of di€erent published studies are hindered by the wide disparity of
analytical methods that have been used, especially as
regards determination of enzyme activities (Dick,
1997). Although total standardization of analytical
methods for biochemical properties may well be impossible because the validity of a given method varies
from one type of soil to another, the development of
biochemical soil quality indices requires, at least, the
availability of data obtained by the same methods for
a signi®cant number of soils of similar characteristics.
The scarcity of comparable data of biochemical
properties is particularly restrictive in the case of
native soils: according to the philosophy of sustainable
development, these soils should be used as standards
for soil quality evaluation because they have developed
freely to attain an equilibrium between their environment and their physical, chemical and biological properties (Doran et al., 1994). What is needed, if
biochemical properties are to realize their potential as
markers of soil quality, is the compilation of comprehensive data bases recording the biochemical properties of native soils in di€erent regions of the world
(Dick, 1997; Pankhurst et al., 1997).
Galicia (NW Spain) is a region of the European
temperate±humid zone in which there still exist many

areas under the climax vegetation, Atlantic oakwood.
The soils of these woodlands are Umbrisols (ISSS
Working Group RB, 1998), and their biochemical
properties have hitherto been studied as little as those
of the native soils of other regions. We have therefore
collected data on a wide range of the biochemical
properties of their organic layers and of the 7 cm of
their Ah horizons. In this paper we summarize our
results concerning general parameters; speci®c parameters, i.e. the hydrolytic enzymatic activities, are the
subject of a companion paper (Trasar-Cepeda et al.,
1999b).

2. Material and methods
2.1. Soils
We studied 40 soils developed under climax vegetation dominated by Quercus robur L. or Q. pyrenaica
L. at sites distributed throughout Galicia, NW Spain
(Fig. 1 and Table 1). All these soils are Umbrisols
(ISSS Working Group RB, 1998); their general physical and chemical properties are summarized in Table 2.
At all sites, the sampling area displayed little disturbance of human origin, and its tree vegetation was composed mainly of healthy mature specimens of the
above-mentioned species.

At each site, samples were taken at 10±15 points distributed uniformly over an area of about 1 ha. After
removal of the litter, samples were taken with a trowel
from the O horizon (which was distinguished by its
morphology and was between 1 and 10 cm deep,
depending on the sampling point) and from the top 7
cm of the Ah horizon. The 10±15 samples for a given
site and layer were pooled in the ®eld to obtain a composite sample representative of that layer at that site.
All samples were collected shortly after the ®rst
autumnal rains, a time of year at which the biochemical properties of these soils are generally close to their
annual means (Trasar-Cepeda et al., 1999a). The O
and Ah pools were transported to the laboratory in
isothermal bags and sieved through 4 mm meshes, and
their moisture contents were determined on the day of
collection prior to storage at ÿ208C pending further
analyses. When withdrawn from the freezer they were
thawed for 1 week at 48C and were maintained at this
temperature until all analyses had been carried out
(always within 15 d).
2.2. Analytical methods
2.2.1. Soil chemical and physical properties

Total C and N contents and pH in KCl were determined following GuitiaÂn and Carballas (1976). Available Ca, Mg and K were extracted with 0.5 M acetic
acid using a soil:extractant ratio of 1:40 and an extraction time of 2 h (GuitiaÂn and Carballas, 1976) and
were determined by atomic absorption spectrometry
(Ca and Mg) or ¯ame spectrophotometry (K). Phosphorus was extracted with 0.5 M sodium bicarbonate
(pH 8.2), and inorganic P was determined by the
method of Murphy and Riley (1962). Amorphous Al
and Fe were extracted with 0.2 M ammonium oxalate/
oxalic acid bu€er of pH 3.4 (McKeague and Day,
1965) and were determined by atomic absorption spectrometry. Particle size distribution was determined
using a Robinson pipette with Calgon as dispersant
(GuitiaÂn and Carballas, 1976). Available water was
determined as the di€erence between the water reten-

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

tions at 33 and 1500 kPa (1/3 and 15 bar, respectively),
which were measured with a Richards plate-and-membrane apparatus using undisturbed soil samples (GuitiaÂn and Carballas, 1976). Table 2 summarizes the
results of these analyses.
2.2.2. Biochemical properties
2.2.2.1. C and N ¯ushes. C and N ¯ushes were determined by the fumigation±extraction method (Vance et

al., 1987), fumigating for 24 h at 258C. The carbon
contents of fumigated and unfumigated samples were
determined by oxidation with potassium dichromate,
and the di€erence was converted to biomass C by

735

dividing by Kec ˆ 0:45: Nitrogen ¯ush was calculated
as the di€erence between ninhydrin-reactive N in fumigated and nonfumigated samples as determined by the
colorimetric method of Joergensen and Brookes
(1990). In each case, C and N were extracted in triplicate and determined in each extract in duplicate;
results shown correspond to the means of all six determinations and are expressed in mg kgÿ1.
2.2.2.2. Soil respiration. Soil respiration was determined by static incubation (GuitiaÂn and Carballas,
1976); the CO2 produced during a 10-d period by 25 g
soil samples incubated at 258C with optimal moisture
content (i.e. the water retained at 33 kPa) was col-

Fig. 1. Map of Galicia with the location of the 40 stands studied.

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

736

lected in 10 mL of a 1 M NaOH solution, which was
then titrated using thymol blue as indicator. Two
samples of each soil were incubated, and the CO2 trap
of each was titrated in duplicate; results shown correspond to the mean of the four values, and to facilitate
comparison with the literature are expressed as mg
CO2-C produced gÿ1 hÿ1, i.e. as the average rate of
CO2 production during the whole 10-d incubation.
2.2.2.3. Nitrogen mineralization capacity. To determine
nitrogen mineralization capacity, duplicate 10 g soil
samples were extracted for 30 min with 50 ml of 2 M
KCl before and after incubation for 10 d at 258C with
optimal moisture content (see Soil respiration), and

ammoniacal N and total inorganic N were determined
in the extracts by Kjeldahl distillation (Bremner, 1965).
Nitrogen mineralization capacity (mg kgÿ1 10 dÿ1) was
calculated from the di€erence between the values

obtained before and after incubation. Results shown
correspond to the mean of two values.
2.2.2.4. Arginine ammoni®cation rate. Arginine ammoni®cation rate was determined by the method described
by Alef and Kleiner (1986). After addition of 0.5 ml of
a 2 g lÿ1 arginine solution to two of four replicate 2 g
soil samples, all four were incubated for 3 h at 308C
and then extracted with 8 ml of 2 M KCl. The ammonium in each extract was determined colorimetri-

Table 1
Location and site characteristics of the soil samples studied
Sample No.

Longitude
(W)

Latitude
(N)

Altitude
(m.a.s.l.)

Slope
(%)

Parent material

Predominant tree species

7
8
10
12
13
14
15
16
17
18
19
20
22
23
24
25
27
28
29
31
33
34
35
36
37
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53

7819'150
7826'100
7818'030
7836'450
7825'220
7856'480
7852'100
8830'400
7812'350
8804'200
8819'300
7835'280
7855'100
7843'510
7808'180
7833'400
8804'100
8841'420
8851'000
9806'220
8846'500
8801'550
8801'380
8827'050
8821'300
8814'150
8809'070
8818'000
8842'510
8832'120
8827'270
8844'330
8806'010
8810'440
8801'050
7851'200
8807'330
8822'490
8839'110
7811'100

42819'400
42821'300
42820'500
41858'580
42806'310
42810'350
42839'380
42836'150
42839'530
42853'000
43801'420
42856'280
42852'450
43815'000
43826'430
43834'550
43825'040
42853'320
42852'290
43800'250
43810'040
43821'200
43824'080
43814'500
42837'000
42822'350
42818'530
42814'400
42807'000
42817'300
42822'410
42828'050
42838'500
42851'150
43802'550
43804'190
43807'100
42858'130
43800'510
43827'180

530
780
830
810
690
370
670
530
1080
400
600
530
460
420
165
350
15
170
255
185
230
380
290
265
665
370
150
660
380
250
400
240
650
335
550
550
540
260
240
580

9
15
33
3
11
18
7
12
50
17
13
5
5
0
22
25
50
13
43
8
23
13
45
0
6
30
29
32
20
15
40
17
10
20
10
7
5
14
25
25

granites
granites
granodiorites
granites
migmatites
schists
granodiorites
granodiorites
slates
slates and quarzites
granodiorites
granites
granodiorites
schists
quarzites and slates
granites
orthogneisses
granodiorites
granodiorites
granites
metagabres
schists
slates and schists
granodiorirtes
schists
granodiorites
granodiorites
granites
granites
granites
granites
granites
schists
schists
paragneisses
granites
orthogneisses
schists
gneisses
quarzites

Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.
Q.

pyrenaica
pyrenaica
robur L.
robur L.
robur L.
robur L.
robur L.
pyrenaica
pyrenaica
pyrenaica
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.
robur L.

L.
L.

L.
L.
L.

Depth of the O horizon
(cm)
2
2
1
2
5
2
3
3
2
2
5
5
4
10
7
3
3
5
7
5
3
5
7
5
5
5
5
7
3
3
5
5
5
3
7
5
5
5
5
4

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

cally by the phenol and sodium nitroprusside method
of Dorich and Nelson (1983), and arginine ammoni®ÿ1 ÿ1
cation rate (mg N-NH+
h ) was calculated as the
4 g
di€erence between the mean value of the two argininetreated samples and the mean value of the two
untreated samples.
2.2.2.5. ATP. To two of four replicate 2 g soil samples
was added 20 ml of a solution of 1 mg of pure ATP in
10 ml of bidistilled water. Each of the four samples
was then extracted for 30 min with 10 ml of a cold solution of 0.5 M trichloroacetic acid and 0.5 M
Na2HPO4 (Jenkinson and Oades, 1979), 100 ml samples
of the ®ltered extracts were added to 900 ml of neutralizing bu€er (0.25 M Tris±HCl+4 mM EDTA, pH
7.5), and ATP was quanti®ed by the luciferine±luciferase method, the light evolved in the 10 s following addition of the enzyme system being determined from
OPTOCOM1 luminometer measurements with the aid
of a calibration line. The results for the spiked samples
and the results for the unspiked samples were then
each averaged. Finally, ATP losses due to sorption by
the soil and degradation during extraction, which were
assumed proportional to original ATP content, were
corrected for using the constant of proportionality calculated from the results for the spiked and unspiked
samples. Results are shown as mg ATP gÿ1.
2.2.2.6. Dehydrogenase activity. Dehydrogenase activity
was determined using a modi®cation of the method of
von Mersi and Schinner (1991) described by CaminÄa et
al. (1998). After addition of 1.5 ml of Tris±HCl bu€er
and 2 ml of a 0.4% solution of INT (2-( p-iodophenyl)-3-( p-nitrophenyl)-5-phenyltetrazolium chloride),

737

triplicate 1 g soil samples were incubated in the dark
for 1 h in a shaking water bath at 408C, mixed
thoroughly with 10 ml of 1:1 ethanol/dimethylformamide, and left at room temperature for 10 min before
®ltration. Iodonitrotetrazolium formation (INTF) was
determined spectrophotometrically in the ®ltrate by
measuring absorbence at 490 nm and reading the corresponding INTF concentration from a calibration line
constructed using samples treated as above except for
the addition of various concentrations of INTF instead
of INT. Results shown (nmol INTF gÿ1 hÿ1) correspond to the mean of the three values.
2.2.2.7. Catalase activity. Catalase activity (EC
1.11.1.6) was determined according to the method of
Johnson and Temple (1964). Triplicate 0.5 g soil
samples were suspended in a mixture of 40 ml of distilled water and 5 ml of 0.3% H2O2. After 10 min stirring at room temperature, 5 ml of 3 M H2SO4 was
added and residual H2O2 was determined by titration
against 0.1 M potassium permanganate. Results shown
(mmol H2O2 consumed gÿ1 hÿ1) correspond to the
mean of the three values.

2.3. Expression and analysis of results
All values reported are expressed on an oven-dry
soil basis (1058C). Statistical analysis were performed
using Statistics 4.5 for Windows (StatSoft, Inc., 1993).
The values, means and standard deviations of the
biochemical parameters measured in the O and Ah
horizons studied are listed in Tables 3 and 4, respectively.

Table 2
Maxima, minima, means and standard deviations (S.D.) of the physical and chemical properties, in the O and Ah horizons of the studied soils
O horizons …n ˆ 40)

pH KCl
Total C (%)
Total N (%)
Available Ca2+ (mg 100 gÿ1)
Available K+ (mg 100 gÿ1)
Labile Pi (mg 100 gÿ1)
Water retained at 1500 kPa (%)
Available water (%)
Al2O3 (%)
Fe2O3 (%)
Sand (%)
Silt (%)
Clay (%)

Ah horizons …n ˆ 40)

minimum

maximum

mean2S.D.

minimum

maximum

mean2S.D.

2.58
14.62
0.77
36
17
19
37
11
0.23
0.11
ND
ND
ND

4.66
49.97
2.17
352
61
124
151
103
1.77
1.94
ND
ND
ND

3.3220.52
30.5328.56
1.5420.35
124270
3629
63225
84226
54218
0.7020.39
0.5920.35
ND
ND
ND

2.77
5.20
0.32
6
6
8
8
11
0.11
0.25
13
12
7

4.10
18.34
0.99
103
24
43
47
132
2.88
1.97
80
60
31

3.4920.32
10.75 123.10
0.6520.18
24220
12214
2029
2924
39221
1.0920.72
0.9320.51
52214
29211
2026

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

738

Table 3
Values of the general biochemical parameters studied, in O horizons …n ˆ 40† of climax soils under oakwood in Galicia (NW Spain)
Sample %
%
Microbial N
CO2-C
qCO2d ATPe Dehydrogenasef Catalaseg NH4
Total
Arginine
Total Total biomass
¯ushb evolvedc
mineralizedh inorganic N ammoni®cationi
C
N
Ca
mineralizedh
7.1
8.1
10.1
12.1
13.1
14.1
15.1
16.1
17.1
18.1
19.1
20.1
22.1
23.1
24.1
25.1
27.1
28.1
29.1
31.1
33.1
34.1
35.1
36.1
37.1
39.1
40.1
41.1
42.1
43.1
44.1
45.1
46.1
47.1
48.1
49.1
50.1
51.1
52.1
53.1

31.2
28.6
14.6
21.2
21.0
18.3
17.3
27.8
23.1
17.8
32.3
26.9
18.2
50.0
46.2
37.8
19.2
36.2
32.4
40.0
28.5
31.0
39.8
39.2
26.6
35.7
36.5
39.3
31.5
39.6
33.4
38.8
26.6
31.0
36.8
38.6
34.5
19.4
25.3
28.8

1.57
1.76
0.95
1.19
1.16
0.97
1.07
1.51
1.29
1.10
1.57
1.56
1.52
1.92
2.07
1.60
1.02
1.66
1.92
2.17
1.50
1.83
1.76
1.83
1.51
1.71
1.83
1.95
1.59
1.86
1.58
1.81
1.26
1.75
1.88
1.80
1.68
0.95
0.77
1.28

1916
3286
1840
1798
1924
2205
1271
2455
1986
1503
2094
1655
1396
2260
1968
1764
1356
1663
2029
2136
1437
1756
1998
2691
1285
1346
2422
2734
2582
1697
2418
2099
1586
1786
1837
2324
1791
1484
2186
1430

43.9
134.6
66.2
47.9
53.6
54.3
40.4
73.7
71.7
52.1
46.6
55.7
43.9
58.2
70.7
54.5
39.7
50.0
81.9
67.5
61.2
74.0
80.5
60.6
90.4
49.6
91.6
101.3
74.9
52.7
90.9
83.8
60.7
91.6
78.6
93.6
68.3
53.5
89.7
74.2

9.7
1.1
8.0
16.4
12.1
6.3
8.7
12.5
14.1
11.7
18.3
12.5
8.6
20.6
18.0
11.0
8.0
13.9
10.1
23.2
10.4
9.4
13.6
17.8
10.5
13.7
24.1
16.5
12.6
11.8
15.8
16.9
12.6
12.6
13.7
12.7
11.9
7.3
11.4
14.2

5.1
3.5
4.4
9.1
6.3
2.9
6.9
5.1
7.1
7.8
8.7
7.6
6.2
9.1
9.2
6.3
5.9
8.4
5.0
10.9
7.3
5.3
6.8
6.6
8.2
10.2
10.0
6.0
4.9
7.0
6.6
8.1
7.9
7.1
7.5
5.5
6.6
4.9
5.2
10.0

6.14
14.05
9.06
11.95
9.64
12.86
9.38
7.10
13.58
5.69
4.69
6.18
3.77
7.87
8.57
8.82
8.57
5.61
5.88
7.68
10.35
10.16
12.07
11.57
7.50
4.62
14.13
6.96
7.88
4.01
11.93
15.26
14.42
11.57
6.03
10.50
12.41
7.21
2.41
8.20

571
1320
657
550
649
722
674
369
923
752
739
786
637
628
636
635
335
544
242
332
471
474
558
347
522
404
494
432
302
398
672
294
413
918
556
398
459
553
471
439

4.4
4.6
7.1
2.2
4.7
2.6
4.4
2.9
6.6
3.0
6.4
5.0
4.9
4.7
3.9
3.8
3.8
3.8
2.4
2.9
3.7
3.5
4.1
5.0
3.1
3.5
4.6
3.4
3.7
2.7
3.4
3.3
4.3
3.1
3.9
2.8
4.0
2.2
3.0
3.7

109
106
22
148
90
ÿ65
46
89
38
84
97
101
74
141
132
74
47
129
108
332
10
6
43
149
29
10
126
152
20
119
171
137
20
125
107
157
88
91
47
101

114
181
52
148
92
ÿ53
56
100
78
84
100
168
78
166
136
85
47
132
157
335
54
73
57
157
36
34
209
152
95
119
210
180
31
147
177
157
117
93
59
110

3.7
13.0
14.5
13.2
10.2
11.3
12.8
8.2
14.2
11.0
7.1
5.0
3.2
0.0
13.0
1.7
10.4
8.3
6.2
10.3
10.6
13.3
13.2
1.8
15.1
8.4
13.8
18.6
6.6
10.5
13.7
12.8
14.4
23.6
26.0
6.3
25.0
8.2
5.9
18.3

Mean
S.D.

30.5
8.6

1.54
0.35

1935
450

68.2
20.0

12.9
4.5

6.9
1.9

8.91
3.29

557
205

3.9
1.1

90
65

113
66

11.1
5.9

a

mg C kgÿ1.
mg N kgÿ1.
c
mg CO2-C gÿ1 hÿ1.
d
mg CO2-C mgÿ1 C biomass hÿ1.
e
mg ATP gÿ1.
f
nmol INTF gÿ1 hÿ1.
g
mmol H2O2 consumed gÿ1 hÿ1.
h
mg N kgÿ1 10 dÿ1.
i
ÿ1 ÿ1
mg N-NH+
h .
4 g
b

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

3. Results and discussion
3.1. Biochemical parameters
3.1.1. Microbial biomass C
In the O horizons, microbial biomass C ranged from
1271 to 3286 mg kgÿ1 (mean 1935 mg kgÿ1, coecient
of variation CV 23%); 38% of the soils had values in
the range 1600±2000 mg kgÿ1. In the Ah horizons,
values ranged from 250 to 1483 mg kgÿ1 (mean 781
mg kgÿ1, CV 32%); most values (63%) were in the
range 600±1000 mg kgÿ1. These data are not dissimilar
to those reported for other soils, for which values in
the ranges 2000±10,000 and 200±1500 mg kgÿ1 have
been reported for O and Ah horizons respectively
(Vance et al., 1987; Sparling et al., 1994; Joergensen et
al., 1995). On average, the proportion of total C content constituted by microbial biomass C was 0.68% in
the O horizons and 0.78% in the Ah horizons; in both
layers, most values lay in the range 0.4±0.6%. These
values are similar to those reported in the literature
(Wardle, 1992).
3.1.2. Microbial biomass N
The fumigation-induced ninhydrin-reactive N ¯ush
was 68.2 2 20.0 mg kgÿ1 (CV 29%) in the O horizons
and 25.6 2 12.6 mg kgÿ1 (CV 49%) in the Ah horizons. These values are within the ranges reported in
the literature (Sparling et al., 1994; Joergensen, 1996).
There was close correlation between microbial biomass
C and the ninhydrin-reactive N ¯ush in both the O
horizons …r ˆ 0:98, P < 0.001) and the Ah horizons
…r ˆ 0:93, P < 0.001); the value of the corresponding
regression coecient, 27 in both cases, is practically
identical to the value of 28.2 reported by Badalucco et
al. (1992).
The literature is not consistent as regards the conversion factor that should be used to transform N
¯ush data into microbial biomass N values. Biomass N
values calculated in this study using a conversion factor of 3.5 (the mean of the 3.10 reported by Amato
and Ladd (1988) and the 3.75 and 3.50 reported by
Sparling et al. (1994)) imply that the proportion of
total N content constituted by microbial biomass N
was 1.60 20.57% in the O horizons and 1.40 2 0.59%
in the Ah horizons. Both ®gures lie towards the lower
ends of the ranges reported in the literature (Wardle,
1992; Joergensen et al., 1995).
3.1.3. Microbial biomass C-to-N ratio
The microbial biomass C-to-N ratio, calculated as
the ratio between the microbial biomass C and microbial biomass N values obtained as above, was 8.52
1.9 for O horizons and 9.8 2 3.2 for Ah horizons. To
calculate the microbial biomass C-to-N ratio directly
from the C and N ¯ushes, total N ¯ush must be esti-

739

mated from ninhydrin-reactive N ¯ush by multiplying
the latter by a factor that, as in the case of the N ¯ush
to biomass N conversion factor, is controversial. Published values include 1.4 (Sparling and Zhu, 1993), 1.8
(Joergensen and Brookes, 1990), to 1.9±2.3 (Inubushi
et al., 1991). In this study the value 1.85 (the mean of
the above) a€orded average microbial biomass C-to-N
ratios of 7.2 for O horizons and 8.3 for Ah horizons;
these values are in acceptable agreement with the
values noted above, suggesting that these two methods
of calculating microbial biomass C-to-N ratios are
equally valid. The fact that for both O and Ah horizons both estimates are greater than 5 shows that in
these soils the microbial biomass is mainly fungal
rather than bacterial, since the C-to-N ratio ranges
from 3 to 5 for bacteria and from 4 to 15 for fungi
(Paul and Clark, 1989).
3.1.4. Soil respiration
CO2 production ranged from 1.1 to 24.1 mg CO2-C
gÿ1 hÿ1 in the O horizons (mean 12.9 mg CO2-C gÿ1
hÿ1, S.D. 4.5 mg CO2-C gÿ1 hÿ1) but were signi®cantly
lower in the Ah horizons, ranging from 1.4 to 5.2 mg
CO2-C gÿ1 hÿ1 (mean 2.6 mg CO2-C gÿ1 hÿ1, S.D. 0.8
mg CO2-C gÿ1 hÿ1). These values are within the
reported ranges for woodland soils (Wardle, 1993;
Gregorich et al., 1994).
3.1.5. Metabolic quotient (qCO2)
The metabolic quotient (the quantity of CO2-C produced per unit of microbial biomass C per unit time;
Anderson and Domsch, 1985) has been considered as
indicative of the maturity of a soil ecosystem, although
severe limitations on its use have recently been stressed
(Wardle and Ghani, 1995). In this study its value was
6.9 2 1.9 in O horizons and 3.5 2 1.3 in Ah horizons.
The Ah values are similar to those reported in the literature (Anderson and Domsch, 1993; Wardle, 1993),
but the O horizons values are considerably greater
than in most reports (Ross and Tate, 1993; PoÈhnacker
and Zech, 1995), although they coincide with the qCO2
values observed by Dilly and Munch (1995) in a study
of the decomposition of Alnus glutinosa (L.) Gaestn.
litter. The fact that in the Ah horizons qCO2 is on
average only about half its value in the O horizon may
be attributed to the lower readily degradable C content
of the Ah horizons, which determines the replacement
of the predominantly zymogenous micro¯ora of the O
horizon by an energetically more ecient autochthonous micro¯ora. Rosenbrack et al. (1995) have
reported that changes in fungal population structure
have similar e€ects on the qCO2 of decomposing A.
glutinosa (L.) Gaestn. litter.
3.1.6. ATP
In the O horizons, 37 2 17% of added ATP was

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

740

Table 4
Values of the general biochemical parameters studied, in Ah horizons …n ˆ 40† of climax soils under oakwood in Galicia (NW Spain)
Sample %
%
Microbial
N
CO2-C
qCO2d ATPe Dehydrogenasef Catalaseg NH4
Total
Arginine
a
b
c
Total Total biomass C ¯ush evolved
mineralizedh inorganic N ammoni®cationi
C
N
mineralizedh
7.2
8.2
10.2
12.2
13.2
14.2
15.2
16.2
17.2
18.2
19.2
20.2
22.2
23.2
24.2
25.2
27.2
28.2
29.2
31.2
33.2
34.2
35.2
36.2
37.2
39.2
40.2
41.2
42.2
43.2
44.2
45.2
46.2
47.2
48.2
49.2
50.2
51.2
52.2
53.2

10.9
10.0
5.7
9.5
6.3
5.2
7.8
12.3
8.3
6.6
12.3
9.5
8.9
14.5
9.5
12.5
11.1
9.4
14.8
11.6
10.3
14.6
12.2
18.3
11.8
11.5
6.6
13.1
9.7
10.2
7.8
15.1
14.7
9.3
13.2
10.1
15.2
6.7
7.9
15.1

0.61
0.70
0.39
0.51
0.37
0.32
0.56
0.76
0.55
0.40
0.65
0.57
0.59
0.81
0.68
0.57
0.54
0.44
0.93
0.79
0.68
0.96
0.77
0.99
0.85
0.74
0.40
0.79
0.63
0.63
0.43
0.85
0.76
0.72
0.92
0.63
0.98
0.41
0.48
0.69

1483
1129
802
695
699
840
690
1020
817
663
589
566
434
832
431
250
606
673
1102
982
600
975
644
1333
567
386
639
858
851
755
682
666
882
786
952
1041
514
740
950
1130

28.3
43.9
19.1
13.6
12.1
15.0
15.6
24.1
14.7
15.8
10.9
13.0
12.5
28.8
15.8
7.7
17.1
19.9
41.2
35.1
24.1
43.4
32.3
32.3
31.0
14.4
18.7
25.2
20.8
26.6
33.7
36.6
31.7
28.7
30.5
27.7
39.8
23.7
24.0
76.5

2.4
3.0
2.4
3.6
1.8
2.2
2.4
2.9
2.8
2.3
2.9
1.9
1.8
3.1
1.6
1.4
1.9
2.2
3.0
2.3
1.7
3.2
2.2
3.6
2.5
3.3
1.9
3.0
2.1
2.6
2.3
3.3
4.6
3.3
2.3
1.9
2.1
2.4
1.4
5.2

1.6
2.6
3.0
5.2
2.5
2.6
3.5
2.9
3.4
3.5
4.9
3.3
4.1
3.8
3.7
5.6
3.1
3.2
2.7
2.4
2.8
3.2
3.4
2.7
4.5
8.5
2.9
3.5
2.5
3.5
3.4
5.0
5.2
4.2
2.4
1.9
4.1
3.3
1.5
4.6

2.40
6.34
1.88
2.06
2.42
6.74
2.98
3.77
1.57
1.42
2.06
4.18
1.40
3.60
2.26
1.46
1.46
2.06
2.72
3.96
2.30
2.24
2.19
4.25
3.86
2.24
4.08
0.82
0.83
2.16
4.29
3.76
2.71
1.87
5.63
1.92
2.90
1.83
1.69
2.31

208
298
185
163
193
96
269
107
340
236
278
235
222
247
213
106
161
216
136
147
162
187
170
117
257
216
198
187
152
170
217
239
307
307
210
174
234
240
211
262

1.9
1.6
1.9
0.8
1.7
0.9
2.5
1.0
3.4
1.1
3.6
2.0
1.7
3.2
1.5
0.8
2.1
1.8
2.0
1.7
3.9
3.3
3.5
2.2
4.2
1.9
0.6
1.9
2.1
1.3
0.6
1.7
3.4
1.6
2.2
1.3
2.2
1.3
1.3
2.9

18
40
19
35
20
1
22
6
11
17
8
16
14
31
23
27
27
28
59
37
8
11
14
39
7
8
12
28
16
26
40
40
21
33
6
18
34
28
18
27

19
55
19
35
20
1
22
21
15
17
11
30
14
31
32
27
27
28
60
69
24
41
35
48
21
15
21
28
43
26
49
54
25
42
47
18
48
33
18
32

4.9
0.9
5.8
5.8
7.0
4.6
6.0
4.1
6.3
1.8
4.4
3.1
3.2
6.3
0.9
1.6
3.5
3.7
8.0
2.5
3.5
5.8
3.8
7.0
3.9
3.8
4.4
3.2
4.8
4.9
6.3
6.7
6.0
7.0
7.2
4.5
8.7
2.7
7.0
11.4

Mean
S.D.

10.8
3.8

0.65
0.18

781
253

25.6
12.6

2.6
0.8

3.5
1.3

2.77
1.38

207
58

2.0
0.9

22
12

30
15

4.9
2.2

a

mg C kgÿ1.
mg N kgÿ1.
c
mg CO2-C gÿ1 hÿ1.
d
mg CO2-C mgÿ1 C biomass hÿ1.
e
mg ATP gÿ1.
f
nmol INTF gÿ1 hÿ1.
g
mmol H2O2 consumed gÿ1 hÿ1.
h
mg N kgÿ1 10 dÿ1.
i
ÿ1 ÿ1
mg N-NH+
h .
4 g
b

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

recovered, and in the Ah horizons 43 2 13%. These
®gures are lower than those reported by Contin et al.
(1995), but similar to those found by Webster et al.
(1984). The lower recovery in the organic layer
suggests that ATP losses were due mainly to hydrolytic
processes mediated by soil biomass, which was greater
in the organic layer than in Ah. The ATP values in the
O and Ah horizons, once corrected for losses, were
8.91 2 3.29 and 2.77 2 1.38 mg gÿ1 respectively. Comparisons with the results of other authors are limited
by the wide variety of methods that have been used
for soil ATP determination, but the amounts found in
this work are similar to those reported by Bolton et al.
(1993) and Contin et al. (1995). It is noteworthy that
the mean ratios between microbial biomass C and
ATP, 220 for O horizons and 280 for Ah horizons, are
close to the values obtained with pure cultures (200±
250; Martens, 1995), which suggests that the microbial
populations of these soils consist predominantly of
active individuals.
3.1.7. Dehydrogenase activity
Dehydrogenase activity was 557 2 205 nmol INTF
gÿ1 hÿ1 in the O horizons (range 242±1320 nmol
INTF gÿ1 hÿ1) and 207 2 58 nmol INTF gÿ1 hÿ1 in
the Ah horizons (range 96±340 nmol INTF gÿ1 hÿ1).
In both cases the measured dehydrogenase activities
had highly symmetric bell-shaped frequency distributions. Comparison with the literature is impossible
given the absence of reported woodland soil dehydrogenase activity data obtained with INT as substrate.
3.1.8. Catalase activity
Catalase activity was 3.9 2 1.1 mmol H2O2 consumed gÿ1 hÿ1 in the O horizons (range 2.2±7.1 mmol
H2O2 consumed gÿ1 hÿ1) and 2.0 2 0.9 mmol H2O2
consumed gÿ1hÿ1 in the Ah horizons (range 0.6±4.2
mmol H2O2 consumed gÿ1 hÿ1). As in the case of dehydrogenase activity, the scarcity of published catalase
data and the diversity of methods used to obtain such
data as have been reported together make meaningful
comparison with the literature impossible.
3.1.9. Nitrogen mineralization capacity
Preincubation inorganic N content ranged from 11
to 235 mg kgÿ1 in the O horizons and from 6 to 74
mg kgÿ1 in the Ah horizons. In both layers, about
90% of inorganic N was ammoniacal. Both the inorganic N contents and the predominance of ammoniacal forms are normal for Galician soils (GonzaÂlezPrieto et al., 1992). Incubation increased total inorganic N content by 113 2 66 mg kgÿ1 in the O horizons and by 30215 mg kgÿ1 in the Ah horizons, with
increases in ammoniacal N (90 2 65 and 22 2 12 mg
kgÿ1, respectively) that show the predominance of
ammoni®cation over nitri®cation in these soils. These

741

values are very similar to those reported in the literature (Ross and Tate, 1993; Gregorich et al., 1994).

3.1.10. Arginine ammoni®cation
The arginine ammoni®cation rate was 11.1 2 5.9 mg
gÿ1 hÿ1 in the O horizons and 4.9 2 2.2 mg gÿ1 hÿ1 in
the Ah horizons. These ®gures are well above the few
values reported in the literature (Alef and Kleiner,
1986; Franzluebbers et al., 1995) and, as with the N
mineralization results reported above, show the high
ammoni®cation capacity of Galician climax soils.

3.2. Correlations between general biochemical
parameters and chemical variables
The above results show that the two layers studied
are very similar in biochemical behaviour, exhibiting
merely quantitative rather than qualitative di€erences.
This suggests, in consonance with the ®ndings of other
authors (Kaiser et al., 1992; Raubuch and Beese,
1995), that these biochemical properties are related to
soil organic matter content. In fact, when the data for
the O and Ah samples are analysed jointly, most of
the general biochemical parameters considered Ð particularly CO2 production, N mineralization capacity
and microbial biomass C and N Ð show close positive
correlation with total C and N contents (Table 5).
This is in keeping with the presence, in these climax
soils, of a balance between biochemical properties and
organic matter content that is re¯ected by both their O
and Ah horizons complying with a single relationship
between total N and certain biochemical properties
(Trasar-Cepeda et al., 1998). The pooled O and Ah
data also show good positive correlation between
nutrients contents and most of the general biochemical
parameters; in particular, it is with nutrient content
that ATP, dehydrogenase and catalase are most closely
correlated (Table 5). Few of the biochemical parameters are signi®cantly a€ected by pH (doubtless due
to the narrow range of variation of this variable in
these soils), and those that are a€ected correlate negatively, as was also found by Dick et al. (1988) and
Joergensen et al. (1995). These negative correlations
are attributed to an indirect e€ect mediated by the accumulation of organic matter. Finally, it may be noted
that neither Al nor Fe oxides correlate highly with any
of the general biochemical parameters studied, and
that such correlations as are signi®cant are negative;
this suggests that the weathering of the parent material, which is the factor chie¯y responsible for the
release of Al and Fe oxides, has little in¯uence on general biochemical properties.

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

±
0.23
±
0.33
0.26
±
0.23
±
±
ÿ0.46
ÿ0.25
±
ÿ0.39
ÿ0.31
ÿ0.23
±
ÿ0.38
±
ÿ0.42
ÿ0.28
±
ÿ0.35
ÿ0.31
±
±
ÿ0.32
±
0.81
0.67
±
0.82
0.63
0.61
0.59
0.72
0.32

% Al2O3
Labile Pi

3.3. Correlations within the general biochemical
parameters

0.87
0.76
±
0.89
0.74
0.77
0.69
0.72
0.50
0.67
0.65
±
0.61
0.69
0.78
0.54
0.51
0.54
0.82
0.80
ÿ0.33
0.88
0.68
0.60
0.61
0.80
0.52
0.81
0.77
ÿ0.27
0.91
0.66
0.55
0.57
0.77
0.47
±
±
±
ÿ0.30
ÿ0.43
±
±
ÿ0.30
±

4. Conclusions

Microbial biomass C
Microbial biomass N
Biomass C/N
CO2-C evolved
ATP
Dehydrogenase
Catalase
Total N mineralized
Arginine ammoni®cation

0.50
0.46
±
0.57
0.31
0.27
0.29
0.36
±

Available K+
Available Ca2+
C/N
% total N
% total C

The mutual correlations among biochemical parameters within each type of layer are weak, apparently
because of the relatively small range of most variables
in each layer. However, when the data for the O and
Ah horizons are analysed jointly, most of the investigated properties are highly correlated (P < 0.001) with
each other (Table 6). In keeping with the ®ndings of
Sparling et al. (1994), Joergensen et al. (1995) and
Franzluebbers et al. (1995), those showing closest correlation with others are in general microbial biomass
C, microbial biomass N and CO2 production. However, in keeping with the observations of Engels et al.
(1993), catalase activity is most closely correlated with
dehydrogenase activity, which together with the close
relationship of both these properties with CO2 production and ATP corroborates the conclusion of Bolton et al. (1993) that both catalase and dehydrogenase
can be regarded as potential markers of soil microbial
activity. This is a potentially useful ®nding in that
both catalase and dehydrogenase are more easily and
rapidly determined than CO2 production, and may
therefore be useful for the rapid estimation of soil microbial activity (Nannipieri et al., 1990). It is also
worth noting that arginine ammoni®cation rate is most
closely correlated with microbial biomass N and ATP,
which shows the relationship between the mineralization of organic matter and both the size and activity
of the microbial population.
As regards parameters derived from biomass C and
N, neither the biomass-C-to-total-C ratio nor the biomass-N-to-total-N ratio exhibit very close correlation
with any of the other variables considered. Nor does
the biomass C-to-N ratio, but it is noteworthy that
this property is negatively correlated with measures of
nitrogen dynamics such as microbial biomass N, N
mineralization capacity and arginine ammoni®cation
rate. Finally, it is also worth noting that qCO2 is closely correlated with most of the primary properties
considered, CO2 production especially, which is in
keeping with the ®ndings of Joergensen et al. (1995).

pH KCl

Table 5
Pearson coecients of correlation between general biochemical parameters and chemical properties (P < 0.001;



P < 0.010;



P < 0.020;



P< 0.050)

% Fe2O3

Available water

742

The data reported above show that in native Galician soils (Umbrisols under climax vegetation of
Atlantic oakwood) the values of general biochemical
parameters vary widely but are generally within previously published ranges for temperate woodland soils
and are much higher in the O horizon than in the Ah
horizon. Derived indices (qCO2, microbial biomass Cto-total C ratio, microbial biomass C-to-ATP ratio,
etc.) suggest that active individuals predominate in the

1
1
0.40
1
0.37
0.38
1
0.68
0.36
0.51
1
0.66
0.55
0.47
0.61
1
0.54
0.51
0.53
0.62
0.47
Microbial biomass C
Microbial biomass N
% Biomass C/total C
% Biomass N/total N
Biomass C/N
CO2-C evolved
qCO2
ATP
Dehydrogenase
Catalase
Total N mineralized
Arginine ammoni®cation

1
0.87
±
0.33
±
0.84
0.49
0.75
0.69
0.60
0.71
0.48

1
±
0.53
ÿ0.52
0.76
0.51
0.74
0.68
0.53
0.69
0.65

1
0.38
0.46
ÿ0.28
ÿ0.54
±
±
±
ÿ0.27
±

1
ÿ0.43
±
±
±
0.25
±
±
0.30

1
±
ÿ0.29
ÿ0.24
±
±
ÿ0.23
ÿ0.38

1
0.85
0.73
0.64
0.63
0.81
0.52

ATP

Dehydrogenase

743

microbial communities of these soils, and that fungi
predominate over bacteria. The fact that qCO2 is signi®cantly lower in the Ah horizons than in the O
layers suggests that the opportunistic organisms typical
of the latter are replaced in the Ah horizons by populations that are better adapted to the soil medium. The
®nding that the general soil properties most closely
correlated with the biochemical properties are the total
C, total N and available nutrient contents suggests
that the number and activity of soil microorganisms
depend mainly on the quantity of mineralizable substrate and the availability of nutrients.

Acknowledgements

qCO2
CO2-C
evolved
Biomass
C/N
% Biomass
N/total
N
% Biomass
C/total
C
Microbial
biomass N
Microbial
biomass
C

Table 6
Pearson coecients of correlation among the general biochemical parameters (P < 0.001;



P < 0.010;



P < 0.020;



P< 0.050)

Catalase

Mineralized
N

Arginine
ammoni®cation

M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000) 733±745

This work was ®nanced by the Xunta de Galicia.
The authors thank Ana Isabel Iglesias-Tojo for her
help with the analysis of the samples.

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M.C. LeiroÂs et al. / Soil Biology & Biochemistry 32 (2000