274 E. Gomez et al. Applied Soil Ecology 15 2000 273–281
ductivity Doran and Parkin, 1996. Soil biological components are fundamental to the development of
ecologically relevant process such as nutrient cycling, improvement of soil structure and xenobiotic decom-
position. Microbial activity dominates the degradation of soil organic substrates and it is of major concern for
ecosystem functioning. Microorganisms are largely sensitive to perturbations. Therefore, changes in the
communities of soil organisms or in their functions may be early signs of alterations in soil health Dick
et al., 1996; Toresani et al., 1998. Maintenance of biodiversity related to soil organisms seems to be cru-
cial for agriculture to be considered sustainable Beare et al., 1995. Nevertheless, studies on diversity have
been focused on higher organisms and only in the recent years more attention is being paid to microor-
ganisms McLaughlin and Mineau, 1995; Kennedy and Gewin, 1997. Some researchers have reported
variable responses of microbial communities to distur- bance. Lupwayi et al. 1998 found that conventional
tillage decreased microbial diversity whereas reduced tillage enhanced it, while Hassink et al. 1991 did not
find differences. Kennedy and Smith 1995 reported greater diversity indexes in cropped systems than in
grasslands when they compared substrate use.
The small size and morphological similarity make the complete morphological and taxonomic charac-
terization of soil microbial communities impossible Garland and Mills, 1991. However, more than the
analysis of taxonomic structure, biochemical and physiological properties may be suitable to a greater
extent from an ecological point of view Hassink et al., 1991; Zak et al., 1994. In this respect, some
authors have pointed out the need of throwing light on the effects of disturbances at a community level
Kennedy and Smith, 1995.
Garland and Mills 1991 applied to whole environ- mental samples a redox technique based in sole car-
bon source utilization profiles, originally designed for strain identification, and found that this assay could
be a useful tool for classifying bacterial communities. Color development due to the reduction of tetrazolium
dye is used as an indicator of catabolism of each car- bon source. Zak et al. 1994 gave this method an
ecological meaning, studying functional diversity by means of indexes of richness, diversity and evenness,
and patterns that emerged from the catabolized carbon sources.
Since then, and despite the successful use of car- bon substrate catabolism in terrestrial and aquatic
ecosystems, recent literature refers to problems con- cerning the Biolog approach, specially as regards
analysis and interpretation of results Insam, 1997; Insam and Hitzl, 1999. Thus, the Bilog system is
considered more adequate for comparisons among microbial communities than for their characteriza-
tion Glimm et al., 1997; Garland and Mills, 1999. Several findings have proved the assay to be useful
to distinguish bacterial communities from differ- ent environmental and soil samples Lehman et al.,
1997; Goodfriend, 1998. However, only a few stud- ies have dealt with the possibility of this method
to show the effects of agricultural intensification on soil.
In this work, we examined whether: a time since clearing and subsequent land use produced different
sole carbon source patterns in the bacterial commu- nities of a vertic soil; or b two sampling depths
showed distinctive substrate catabolism profiles. To as- sess whether relationships among bacterial communi-
ties responded to years since clearing and subsequent management, patterns of potential substrate utilization
were compared using the same soil, but in its native condition as reference, using the approach mentioned
by Parkin et al. 1996.
2. Materials and methods
2.1. Sample collection and conditioning This work is part of a Natural Resources Conserva-
tion Project of an area of vertic soils in Entre R´ıos, Ar- gentina 31
◦
30
′
S latitude; 59
◦
45
′
W longitude. Sites for the study were selected on the basis of time elapsed
since native vegetation clearing and management his- tory. Samples from four locations S1, S2, S3, S4 on
a same soil type were collected with a core 2.5-cm diameter at two depths 0–7.5 and 7.5–15 cm on 28
October 1998. Table 1 describes the main character- istics of sampling sites. The soil was a Vertic Argiu-
doll fine, montmorillonitic, thermal family with clay, 236 g kg
− 1
, and silt, 737 g kg
− 1
, in the Horizon A. Table 2 shows the measurements of organic carbon
OC, carbon:nitrogen ratio CN and pH for each lo- cation in both the sampling depths.
E. Gomez et al. Applied Soil Ecology 15 2000 273–281 275
Table 1 Description of sampling sites: the native condition, and the other sites selected on the basis of time elapsed since native vegetation clearing
and management history Location 1 S1
Native vegetation: xerophytic bush Prosopis sp., Celtis sp. and herbaceous Stipa, Setaria, Bothriochloa, Paspalum, Stenandrium, Scoparia, Trifolium
Location 2 S2 16 years since clearing; cropped by the first 8 years with conventional tillage moldboard
plowing as the main labor; with naturalized prairie Bromus sp. since 1990 Location 3 S3
26 years since clearing; continual cropping with corn Zea mays L. and soybean Glycine maxL. Merr under conventional tillage; moldboard plowed a week before sampling
Location 4 S4 40 years since clearing; continual cropping with corn and soybean; managed with zero tillage
since 1994; soybean residue covering at sampling
In order to evaluate internal variability, plots of each location were divided into sectors 50 m
2
. Twenty sub-samples from each sector were composited. Three
replicates from each location were sieved 2 mm, stored at 4
◦
C and processed within a week. 2.2. Sample analysis
The Biolog Gram negative GN microplates Bi- olog Inc. Hayward CA, 1993 were used. The Biolog
GN and Gram positive GP microplates are not selec- tive for either GN or GP bacteria Zak et al., 1994.
Most of the substrates in GN plates are also found in GP plates. It has been argued that several of the
C-sources in Biolog plates are not present in ecosys- tems Konopka et al., 1998. We chose working with
GN plates, since some of the C-sources found in GN, but not in GP, microplates have been reported as con-
stituents of root exudates Campbell et al., 1997. Each plate consists of 96 wells 95 with a carbon source
incorporated to a basal medium as sole C-source and one control without any C-source. Tetrazolium violet
is used as a redox dye to colorimetrically indicate the utilization of the carbon sources. Carbon compounds
Table 2 Organic carbon OC, carbon:nitrogen ratio CN and pH in water 1:2.5 measured in soil from the sampling sites at depths of 0–7.5
and 7.5–15 cm, respectively Sampling sites
OC
a
CN
b
pH 0–7.5 cm
7.5–15 cm 0–7.5 cm
7.5–15 cm 0–7.5 cm
7.5–15 cm S1
5.53 3.92
12.86 13.52
6.2 6.0
S2 5.11
3.47 11.88
11.19 6.2
6.1 S3
2.40 2.18
11.43 10.90
6.2 6.0
S4 2.64
2.73 13.20
13.64 6.4
6.3
a
Walkley–Black method.
b
Kjeldahl method.
included in Biolog GN microplates and grouped into guilds by chemical structure Zak et al., 1994 are
shown in Table 3. Soil suspensions from each bulked sample soil
10 g; sterile saline 0.85 NaCl 100 ml were shaken for 1 h and pre-incubated for 18 h before inoculation
to allow microbial utilization of any soluble organic C derived from the soil Dick et al., 1996. Ten-
fold dilutions were performed and aliquots of 100 ml from 10
− 4
dilution were inoculated into each well of the Biolog GN microplates. The plates were in-
cubated at 25
◦
C. Color development in each well was recorded as optical density OD at regular time
intervals with an MR700 Dynatech Plate Reader at 590 nm.
Bacterial plate counts onto tryptic soy agar TSA were also performed for each sample after 5 days
of incubation at 25
◦
C, and results were expressed as colony-forming units CFU g
− 1
soil. 2.3. Data analysis
Microbial activity in each microplate, expressed as average well-color development AWCD was deter-
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
