276 E. Gomez et al. Applied Soil Ecology 15 2000 273–281
Table 3 Carbon compounds included in the Biolog GN microplates
a
Carbohydrates Adonitol 8
d -Mannitol 22
i-Erythritol 12 Mono-methyl-succinate 35
a -d-Glucose 17
d -Mannose 23
l -Arabinose 9
N-acetyl-d-galactosamine 6 a
-d-Lactose 19 d
-Melibiose 24 l
-Fucose 14 N-Acetyl-d-glucosamine 7
b -Methyl-glucoside 25
d -Raffinose 27
l -Rhamnose 28
Sucrose 30 Cellobiose 11
d -Psicose 26
Lactulose 20 Turanose 32
d -Arabitol 10
d -Sorbitol 29
m-Inositol 18 Xylitol 33
d -Fructose 13
d -Trehalose 31
Maltose 21 d
-Galactose 15 Gentiobiose 16
Methyl pyruvate 34 Carboxylic acids
Acetic acid 36 cis-Aconitic acid 37
d -Glucosaminic acid 43
Malonic acid 54 a
-Hydroxybutyric acid 45 Citric acid 38
d -Glucuronic acid 44
p-Hydroxy-phenylacetic acid 48 a
-Ketobutyric acid 50 d,l
-Lactic acid 53 d
-Saccharic acid 57 Propionic acid 55
a -Ketoglutaric acid 51
d -Galactonic acid lactone 40
Formic acid 39 Quinic acid 56
a -Ketovaleric acid 52
d -Galacturonic acid 41
g -Hydroxy-butyric acid 47
Sebacic acid 58 b
-Hydroxybutyric acid 46 d
-Gluconic acid 42 Itaconic acid 49
Succinic acid 59 Amino acids
d,l -Carnitine 82
Glycyl-l-glutamic acid 71 l
-Aspartic acid 68 l
-Phenylalanine 76 d
-Alanine 64 Hydroxy-l-proline 73
l -Glutamic acid 69
l -Proline 77
d -Serine 79
l -Alanine 65
l -Histidine 72
l -Pyroglutamic acid 78
g -Aminobutyric acid 83
l -Alanyl-glycine 66
l -Leucine 74
l -Serine 80
Glycyl-l-aspartic acid 70 l
-Asparagine 67 l
-Ornithine 75 l
-Threonine 81 Amines-Amides
Polymers 2-Amino-ethanol 90
Phenyl-ethylamine 88 a
-Cyclodextrin Tween 40 4
Alaninamide 63 Putrescine 89
Dextrin 2 Tween 80 5
Glucuronamide 62 Succinamic acid 61
Glycogen 3 Others
2,3-Butanediol 91 Glucose-6-phosphate 95
Thymidine 87 Bromosuccinic acid 60
Glycerol 92 Uridine 86
d,l -a-glycerolphosphate 93
Inosine 85 urocanic acid 84
Glucose-1-phosphate 94
a
C substrates are numbered as they are located in the GN microplates.
mined as follows for the different measurement times: AWCD =
P OD
i
95 where OD
i
is the optical density value from each well. Substrate richness R was determined as the total of
reactions in each microplate, considering as ‘positive’ — that is the substrate was metabolized — those wells
with OD0.10 Garland, 1997. Diversity Shannon’s index H was calculated as follows Zak et al., 1994;
Derry et al., 1998
H = − X
p
i
ln p
i
where p
i
is the ratio of the activity on each substrate to the sum of activities on all substrates.
Optical density values OD corresponding to 54 h after inoculation were used to do statistical analysis
SAS, version 6.12. The ANOVA was performed for AWCD, richness, and diversity, considering the effects
from sectors, depths, locations and their interactions. Principal component analysis PCA was used to study
patterns of C-sources catabolism.
Plate counts values were log-transformed for the analysis.
3. Results
3.1. Plate counts and microbial activity In the analysis of CFU g
− 1
of soil, the interac- tion between locations and depths was significant
E. Gomez et al. Applied Soil Ecology 15 2000 273–281 277
Table 4 Bacterial counts, and average color development AWCD, richness and diversity calculated on data from the 54 h of incubation of Biolog
GN microplates, for the native conditions S1, 16-year-clearing location S2, 26-year-clearing location S3 and 40-year-clearing location S4
Sampling sites
Bacterial counts CFU g
− 1
soil AWCD
Richness Diversity
Shannon’s index 0–7.5 cm depth
S1 1.5×10
7
b
a
0.81 a 94 a
4.39 a S2
1.8×10
7
b 0.49 b
81 b 4.23 b
S3 2.6×10
7
b 0.74 a
85 b 4.28 b
S4 1.6×10
8
a 0.37 c
61 c 4.06 c
7.5–15 cm depth S1
5.5×10
7
ns
b
0.78 a 94 a
4.36 a S2
5.3×10
7
ns 0.71 ab
87 ab 4.24 b
S3 2.3×10
7
ns 0.69 b
88 ab 4.31 a
S4 4.3×10
7
ns 0.53 c
82 b 4.24 b
C.V. 2.81
5.03 3.84
0.67
a
Within a column, means followed by the same letter are not significantly different Duncan; p0.01.
b
Not significant.
p0.01. When each depth was considered sepa- rately, significant differences p0.01 were observed
only in the depth of 0–7.5 cm. The 40-years-clearing samples S4 were different from the other locations
Table 4.
The highest AWCD values for the different mea- surement times were encountered in samples from
the native condition S1 while samples from S4 had the lowest values at both these depths Fig. 1. All of
the effects tested by ANOVA sectors, depths, loca- tions and their interactions were significant p0.01
for the AWCD from the 54-h incubation time. The highest values for both depths were encountered in
samples from the native condition S1, but it was not significantly different from the 26-year clearing sam-
ples S3 at depths of 0–7.5 cm. The AWCD value from S2 was lower than in S1 and S3 in the top layer
and was between S1 and S3 at depths of 7.5–15 cm. The lowest AWCD for both these depths were found
in S4 despite the fact that it had the largest number of bacteria in the top 0–7.5 cm layer Table 4.
3.2. Richness and diversity indexes The interaction between locations and depths was
significant in both richness and diversity indexes p0.001, and also locations studied at each depth
of sampling were different.
Fig. 1. Bacterial activity measured by optical density and expressed by average well color development, for pristine location S1,
16-year-clearing location S2, 26-year clearing location S3, and 40-year-clearing location S4, in the two sampling depths 0–7.5
and 7.5–15 cm.
278 E. Gomez et al. Applied Soil Ecology 15 2000 273–281
Fig. 2. Principal component analysis on the 95 C-source absorbance data from 54-h incubation time, for samples from S1, S2, S3 and
S4 at the depth of 0–7.5 cm.
Richness and diversity were largest in S1 for both these depths, though S2 and S3 did not significantly
differ in richness from S1, and S3 did not differ in diversity from S1 at depths of 7.5–15 cm. The lowest
values in both these indexes were found in S4 at depths of 0–7.5 cm Table 4.
3.3. Patterns of substrate utilization At depths of 0–7.5 cm, PCA on data corresponding
to 54 h of incubation allowed the separation of samples from the different locations with 63.3 of the variance
explained by both the first PC1 and second PC2 principal components. Based on C-source usage, S2
and S4 were differentiated from S1 and S3 by PC1 Fig. 2. The 95 C-sources were plotted in a factorial
axes system and their positions indicate the correlation with PC1 and PC2 Fig. 3.
In S2 and S4, there was a lower activity on all the carbon sources than in S1 and S3, with the exception of
substrates 38, 57 and 59 citric acid, D-saccharic acid and succinic acid, respectively, of which high con-
sumption in S2 and S4 allowed the separation Fig. 3.
In respect to S1 and S3, there was a high activity on most substrates in both of them, but CP2 separated
them on the basis of predominance of certain chemical structures that were metabolized by S1 or S3.
Principal components separated samples from the four locations at depths of 7.5–15 cm, but in a some-
what different way, with 43.5 of the variation ex- plained by both PC1 and PC2. The first PC separated
S4 from the other locations, while S2 clustered near samples from S1. As in the case of 0–7.5 cm depths,
Fig. 3. Scores of the 95 C-substrates determined by their correlation with PC1 and PC2 in the PCA on absorbance data from the 54-h
incubation time, for samples S1, S2, S3 and S4 at the depth of 0–7.5 cm.
Fig. 4. Principal component analysis on the 95 C-source absorbance data from 54-h incubation time, for samples from S1, S2, S3 and
S4 at the depth of 7.5–15 cm.
Fig. 5. Scores of the 95 C-substrates determined by their correlation with PC1 and PC2 in the PCA on absorbance data from the 54-h
incubation time, for samples S1, S2, S3 and S4 at the depth of 7.5–15 cm.
E. Gomez et al. Applied Soil Ecology 15 2000 273–281 279
Table 5 Carbon substrates in the Biolog GN microplates that were not
used by one or more locations at the 0–7.5 cm depth S1
S2 S3
S4 Carbohydrates
Adonitol 8 +
a
−
b
+ −
a -d-Lactosa 19
+ +
+ −
b -Methyl-glucoside 25
+ +
+ −
Cellobiose 11 +
+ +
− d
-Melibiose 24 +
+ +
− d-
Raffinose 27 +
+ +
− Gentiobiose 16
+ −
+ −
i-Erythritol 12 +
+ +
− l
-Fucose 14 +
− +
− Lactulose 20
+ +
− −
N-Acetyl-d-galactosamine 6 +
+ +
− Turanose 32
+ −
+ −
Xylitol 33 +
+ −
− Carboxilic acids
a -Hydroxybutiric acid 45
− −
− −
a -Ketobutyric acid 50
+ −
− −
a -Ketovaleric acid 52
+ −
− −
Formic acid 39 +
+ +
− Itaconic acid 49
+ −
+ −
Sebacic acid 58 +
− −
− Amino acids
d -Serine 79
+ +
+ −
Glycyl-l-aspartic acid 70 +
+ +
− Hydroxy-l-proline 73
+ +
+ −
l -Hystidine 72
+ +
+ −
l -Ornithine 75
+ +
+ −
l -Phenylalanine 76
+ +
+ −
l -Threonine 81
+ −
+ −
Aminesamides 2-Amino-ethanol 90
+ −
+ −
Alaninamide 63 +
− −
− Phenyl-ethylamine 88
+ −
+ −
Succinamic acid 61 +
+ +
− Polymer
a -Cyclodextin 1
+ −
+ −
Others 2,3-Butanediol 91
+ −
− −
Thymidine 87 +
+ +
−
a
Indicates OD0.10.
b
Indicates OD0.10.
PC2 differentiated S1 from S3 Fig. 4. More sub- strates than in the surface layer of 0–7.5 cm were me-
tabolized by S2 and S4 in the 7.5–15 cm layer. Mi- crobial communities from S4 assimilated sources 38,
57 and 59 with a high intensity, as they did in the top layer Fig. 5.
Table 6 Carbon substrates in the Biolog GN microplates that were not
used by one or more locations in the depth of 7.5–15 cm S1
S2 S3
S4 Carbohidrates
Adonitol 8 +
a
−
b
+ −
N-Acetyl-d-galactosamine 6 +
− +
+ Xylitol 33
+ +
+ −
Carboxylic acids a
-Hydroxybutyric acid 45 −
− −
− a
-Ketobutyric 50 +
− −
− a
-Ketovaleric acid 52 +
+ −
− Amino acids
l -Ornithine 75
+ +
+ −
l -Phenylalanine 76
+ +
− +
l -Threonine 81
+ −
− +
AminesAmides Alaninamide 63
+ −
− −
Phenyl-ethylamine 88 +
+ −
− Others
Thymidine 87 +
+ +
−
a
Indicates OD0.10.
b
Indicates OD0.10.
The consumption in the amino acid and amineamide guilds seemed to better explain the differences among
S1 and S3. The following substrates were used by S3 at a lower intensity 0.1OD0.25 than by
S1: glycil-l-aspartic acid, glycil-l-glutamic acid, l
-leucine, l
-ornithine, l
-phenilalanine, d
-serine, l
-threonine, 2-amino-ethanol and glucuronamide. Communities from native condition S1 metabo-
lized all the substrates except a-hydroxybutyric acid OD0.10. There were some of the C-sources that
were not used by any of the other locations at both depths. In the 0–7.5 cm deep layer, the largest number
of C-sources that were not assimilated was found in S4. There were less substrates that were not used in
the 7.5–15 cm layer, but the response in the activity on them was more variable among the locations than
in the top layer Tables 5 and 6.
4. Discussion
