Soil Biology & Biochemistry 32 (2000) 1877±1883

www.elsevier.com/locate/soilbio

Soil microbial activity as a biomarker of degradation and

remediation processes
J.A. Pascual*, C. Garcia, T. Hernandez, J.L. Moreno, M. Ros
Department of Soil and Water Conservation and Organic Waste Management, CEBAS-CSIC, P.O. Box 4195, E-30080, Murcia, Spain
Accepted 4 May 2000

Abstract
Several organic matter fractions together with biological and biochemical parameters were measured in a range of intensively farmed soils
in SE Spanish Mediterranean region, which had been abandoned (i.e. not used in agriculture) for different periods of time. These soils were
compared with adjacent natural soils that had never been used for agriculture. There was a general decline of total organic carbon (TOC),
extractable humic substances, water-soluble carbon (WSC) and carbohydrates, microbial biomass and respiration with the time elapsed since
abandonment. There was also a decline in plant cover in the abandoned soils. When a degraded soil was amended with municipal solid waste
at rates of 6.5 and 26 kg m 22 as a potential means of remediation, TOC, humic substances, WSC, microbial biomass and respiration rates
signi®cantly increased but only at the higher rate of amendment. Plant cover was signi®cantly enhanced by both rates of the amendments and
was still present 10 years after the amendment. These data con®rm that agricultural soil abandonment leads to soil degradation and that the
addition of urban waste could be a suitable technique with which to restore their quality. q 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Soil remediation; Organic matter; Dehydrogenase; Hydrolases

1. Introduction the effects of the semiarid conditions (Garcia et al., 1996)

Soil is an important natural resource that needs to be
preserved and, if possible, its quality and productive capa-
city improved. Doran and Parkin (1994) de®ned soil quality
as ªthe capacity to function within an ecosystem and sustain
biological productivity, maintain environmental quality and
promote plant, animal and human healthº. In natural condi-
tions, soils tend towards maintaining an equilibrium
between pedogenetic properties and the natural vegetation
(Parr and Papendick, 1997).
Soil equilibrium can easily be disturbed, especially by
human intervention (e.g. unsuitable agricultural practices).
Furthermore, in the Mediterranean region of SE Spain inap-
propriate agricultural practices are compounded by adverse
environmental and climatic factors (LoÂpez BermuÂdez and
Albaladejo, 1990). Soils from semi-arid regions are not resi-
lient to the effects of inappropriate land-use and manage-
ment, which leads to permanent degradation and loss of
productivity. A key factor in degradation of these soils is
the loss of natural plant cover, allowing soil water erosion
and salinisation processes to occur. This further aggravates
* Corresponding author. Fax: 134-968-266613.
E-mail address: recsuelo@natura.cebas.csic.es (J.A. Pascual).
0038-0717/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0038-071 7(00)00161-9
and leads to a loss of soil quality and fertility and the subse-
quent abandonment of the land for crop production
purposes.
One method of reversing the degradation that is taking
abandoned soil back into agricultural production and
improving the quality of soils with low organic matter
content, involves the addition of municipal solid wastes
(Pascual et al., 1998). These materials are rich in carbon
and energy sources that increase the soil microbial popula-
tion and its activities, and thus reactivate biogeochemical
nutrient cycles (Pascual et al., 1997). The organic wastes
also increase the soil water-holding capacity, aggregation
and improve nutrient status.
Many properties must be used to de®ne soil quality and,
once these have been quanti®ed, the most suitable strategies
for soil management can be undertaken. Chemical and
physical soil parameters such as organic matter, nutrient
status, run-off measurements or aggregate content have
been used to measure soil quality (Parr and Papendick,
1997). However, these parameters change very slowly,
and therefore many years are required to measure signi®cant
changes. On the other hand, soil biological and biochemical
properties are responsive to small changes that occur in soil,
thereby providing immediate and accurate information on
1878 J.A. Pascual et al. / Soil Biology & Biochemistry 32 (2000) 1877±1883

Table 1 2. Materials and methods

Characteristics of abandoned agricultural soils and natural soils. (WHC:
Water holding capacity, EC: electrical conductivity, TOC: total organic
2.1. Study of degraded, abandoned agricultural soils
carbon, means are indicated ^ standard deviation)

Time elapsed since abandonment agricultural Abandoned agricultural soils from the SE Spanish Medi-
terranean region, within the province of Murcia were
,10 years 10±20 years .20 years Natural soil
studied. In an area measuring approximately 4 km 2, 12
Texture type Clay loam Clay loam Clay loam Clay loam sampling sites were chosen to cover intensively farmed
WHC (%) 37.2 ^ 2.1 37.5 ^ 1.5 34.6 ^ 3.8 46.0 ^ 3.0 agricultural soils abandoned for different lengths of time.
PH (1:10) 8.58 ^ 0.2 8.22 ^ 0.31 7.5 ^ 0.1 7.76 ^ 0.9 The soils were grouped according to the time elapsed
EC (dS m 21) 0.69 ^ 0.05 0.79 ^ 0.51 0.83 ^ 0.10 0.25 ^ 0.10 since abandonment (,10, 10±20 and . 20 years). The data
TOC (g kg 21) 11.01 ^ 2.33 6.22 ^ 0.72 5.30 ^ 0.67 20.2 ^ 5.81
referring to the dates when the ®elds were abandoned were
provided by the landowners. The areas were sampled in May
of 1997. All of the soils were clay loams and they were all
changes in soil quality. This is because soil microbial activ- exposed to the same semi-arid climate (rainfall , 250
ity has a direct in¯uence in a ecosystem stability and fertility mm yr 21; annual average temperature 178C).
(Smith and Papendick 1993). Microorganisms play a funda- To ascertain how the soils studied differed from others
mental role in establishing biogeochemical cycles and are from the same area that had not been subjected to human
involved in forming the structure of a soil (Harris and Birch, intervention, a control site supporting natural vegetation
1989). typical from Mediterranean soils (principally Quercus
In this paper, the changes in soil quality taking place in rotundifolia) was included.
agricultural soils, in semiarid conditions at different times Three samples were taken from each of the sampling
after abandonment were evaluated by comparison with sites: each sample consisted of eight subsamples taken
natural soils exposed to the same climate but not from the top 15 cm of soil. The subsamples were mixed,
subjected to intensive agriculture and subsequent aban- homogenised, sieved (,2 mm) and stored at 48C until
donment. We also report results obtained from a ®eld analysed. The main characteristics of the soils are shown
site abandoned 20 years previously and amended with in Table 1.
the organic fraction of a municipal solid waste (MSW)
10 years previous to this study. To monitor soil quality, 2.2. Long-term soil remediation after the addition of the
organic matter fractions (total organic carbon, humic organic fraction of a municipal solid waste
substances, water-soluble carbon and carbohydrates),
microbiological (microbial biomass C, basal respiration) A soil from an area that had been abandoned for 20 years
and biochemical (dehydrogenase, phosphatase, b-gluco- was amended with the organic fraction of a municipal solid
sidase, urease and protease activity) properties were waste (MSW) from Murcia (6.5 and 26 kg m 22). The
measured. organic matter was incorporated into the top 15 cm
using a rotovator. Three plots (one for each treatment
and a control) were set up on an east-facing hill slope
(10% gradient) with a 40-m 2 size. Soil was sampled
Table 2
from each plot 10 years after the amendment. For
Characteristics of the soil and the municipal solid waste use in the soil sampling, eight subsamples were taken randomly from
remediation experiment the top 15 cm of soil, mixed and sieved (,2 mm)
before analysis. The main characteristics of the soil
Soil Municipal solid waste
and MSW are shown in Table 2.
pH (H2O) 7.7 6.8
Electrical conductivity (S m 21) 0.78 5.20
2.2.1. Analytical parameters
Total organic carbon (g kg 21) 5.41 300.1
Humic substances (g kg 21) 1.20 32.3 The total organic C (TOC) content was determined by
Total nitrogen (g kg 21) 0.41 13.1 oxidation with K2Cr2O7 in a concentrated H2SO4 medium
Total phosphorus (g kg 21) 0.58 5.6 and measurement of the excess dichromate using
Total potassium (g kg 21) 8.10 3.2 (NH4)2Fe(SO4)2 (Yeomans and Bremner, 1989). Humic
Water holding capacity (%) 34.9 ±
substances extracted with pH 9.8, 0.1 M sodium pyropho-
Texture type Clay loam ±
Cu (mg kg 21) ,0.1 233 sphate (solid±liquid ratio 1:10) and water-soluble carbon
Zn (mg kg 21) ,0.1 600 extracted with distilled water (1:5 solid±liquid ratio) were
Cr (mg kg 21) ,0.1 345 determined by oxidation with K2Cr2O7 and measurement of
Cd (mg kg 21) ,0.1 3 absorbance at 590 nm (Sims and Haby, 1971). Soluble
Ni (mg kg 21) ,0.1 289
carbohydrates from the water extract were determined by
Pb (mg kg 21) ,0.1 221
the method of Brink et al. (1960).
 J.A. Pascual et al. / Soil Biology & Biochemistry 32 (2000) 1877±1883
Fig. 1. Total organic carbon (TOC), humic substance carbon (A), water
soluble carbon (mg C kg 21 soil) and water-soluble carbohydrates (mg
glucose kg 21) (B) in natural and abandoned agricultural soils at different
times since abandonment. (Error bars denote standard deviation;
least signi®cant difference at P # 0:05; TOC ˆ 1:20; humic
substance carbon ˆ 0.18, water-soluble carbon ˆ 15.0, water-soluble
carbohydrates ˆ 12.0).
Microbial biomass C was determined by fumigation±
extraction method (Vance et al., 1987), after oxidation
with K2Cr2O7, the C content was measured at 590 nm
(Sims and Haby, 1971). Soil respiration was determined
using 50 g dry soil, moistened to 65% of its water-holding
capacity, placed in hermetically sealed ¯asks and incubated
for 30 d at 288C. The CO2 emitted was periodically
collected in 10 ml 0.1 M NaOH and titrated with 0.1 M
HCl (Parr and Smith, 1969).
Dehydrogenase activity was determined by the reduction
of 2-p-iodo-3-nitrophenyl-5-phenyl tetrazolium chloride to
iodonitrophenylformazan by the method of Skujins (1976)
as modi®ed by Garcia et al. (1993). Urease and protease (as
N-a-benzoyl-l-argininamide (BAA) protease) activity were
measured following the method of Nannipieri et al. (1980).
1879
Phosphatase and b-glucosidase activities were determined
using p-nitrophenyl phosphate disodium (0.115 M) and p-
nitrophenyl glucopyranoside (0.05 M), respectively, as
substrates (Tabatabai, 1982).
Biomass C, CO2-C emission, dehydrogenase and hydro-
lases activities were determined immediately after
sampling, while the other analysis were carried out after
storage at 48C for less than 30 d. All assays were carried
out by triplicate and data were analysed statistically, using
the Statgraph version 4.1 software program.
3. Results and discussion
3.1. Degraded, abandoned agricultural soils
Plant cover is an important soil quality factor (Brockway
et al., 1998), mainly due to its contribution towards main-
taining a stable biological population in soil by supplying
carbon and energy sources from root exudates and plant
remains (Balloni and Favilli, 1987). The percentage of
plant cover was estimated by a grid-line intersect method.
The plant cover supported by the abandoned soils were 5%
(,10 years abandonment) and ,2% (10±20 and . 20 years
abandonment). However, the natural soils showed 60%
plant cover, supporting natural vegetation typical from the
area, mainly Quercus rotundifolia. Natural plant re-estab-
lishment could be expected after abandonment of agricul-
tural management (Garcia et al., 1997) but it did not occur
here due to the low level of organic matter, low microbial
activity and extreme climatic conditions (i.e. very long dry
periods).
Total organic carbon (TOC) of the degraded soils ranged
from 4.40 to 12.90 g kg 21 (Fig. 1A), with many soils having
values ,10.00 g kg 21. The TOC content of the abandoned
soils was below that of the natural soil, since agriculture
favours organic matter mineralisation (Tate, 1987), and
this may result in dif®culties for plant establishment after
soil abandonment. TOC decreased with the time, con®rming
the continuing degradation of the soil after abandonment.
The humic carbon content of abandoned soils ranged
from 0.67 to 2.33 g kg 21, values signi®cantly lower than
those of the natural soils (Fig. 1A). The arid climatic condi-
tions to which these soils are exposed and the consequent
slow rate of humi®cation could be the main factor contribut-
ing to these low levels. As with TOC, the lowest values of
humic C were in the soils abandoned for the longest times,
whereas those abandoned less than 10 years ago had signif-
icantly higher humic C contents.
The water-soluble fraction of soil organic matter is of
special importance because it is the most degradable, acting
as an immediate energy source to the microorganisms (Cook
and Allan, 1992). The study of this fraction is also of interest
in agricultural soils because it determines the activity of the
soils (Janzen et al., 1992). The abandoned soils contained
considerably lower levels of water-soluble carbon than did
1880 J.A. Pascual et al. / Soil Biology & Biochemistry 32 (2000) 1877±1883

processes and the low rainfall (LoÂpez BermuÂdez and Alba-

Fig. 2. Microbial biomass carbon (A) and basal respiration (B) in natural
and abandoned agricultural soils at different time since abandonment.
(Error bars denote standard deviation; least signi®cant difference at P #
0:05; microbial biomass carbon ˆ 56.0, basal respiration ˆ 3.2).
the natural soils, which have retained their cover of Quercus
rotundifolia (Fig. 1B). The water-soluble carbon content
declined with time after abandonment, due the continuous
degradation of soil quality as consequence of the erosion
ladejo, 1990). In the abandoned soils, the water-soluble
carbohydrates, which represent the most mineralisable frac-
tion of the organic matter (De Luca and Keeney, 1993), also
had considerably lower levels than in natural soils, but they
showed no signi®cant difference with elapsed time (Fig.
1B).
Throughout this study, the time elapsed since soil aban-
donment seems to be an important factor in¯uencing
organic matter fractions. After intensive agriculture, soils
are often exhausted (Tate, 1987), and if they are abandoned
without any subsequent treatment, they may be subjected to
erosion (Garcia et al., 1996), due to their low capacity to
recover and to establish a natural plant cover.
Microbial biomass C can be considered to be a more
sensitive indicator of soil quality than organic matter or
TOC, since it responds more rapidly and to a greater extent
to changes (e.g. degradation; Ross et al., 1982; Powlson et
al., 1987). The microbial biomass C detected in the aban-
doned soils varied greatly but it declined when agricultural
soils were abandoned and decreased with time elapsed (Fig.
2A), presumably as a consequence of the loss of the capacity
to protect soils against the erosion processes.
Basal respiration is a good indicator as soil microbial
activity (Anderson, 1982) The basal respiration in all the
abandoned soils showed signi®cantly lower values than in
natural soils (Fig. 2B). The lowest values for this parameter
were in the soils abandoned for the longest times.
Dehydrogenase activity has been proposed as a measure
of microbial activity in soil (Garcia et al., 1993), although
some authors have criticised this approach (Nannipieri et al.,
1990; Beyer et al., 1992) because the enzyme is affected by
numerous factors (soil type, pH, etc). Dehydrogenase activ-
ity is involved in the initial breakdown of soil organic matter
(Bolton et al., 1985). We found that the abandoned soils,
which showed low values for other measures of microbial
activity (e.g. biomass carbon content, basal respiration rate),
also display the lowest dehydrogenase activity (Table 3). In
general, the lowest values for dehydrogenase activity were
in the soils abandoned for the longest time, whereas those
abandoned less than 10 years had signi®cantly …P # 0:05†
higher contents. The decrease in activity with the passing
time may be due to progressive erosion of the abandoned

Table 3
Dehydrogenase and hydrolase enzyme activities in natural soil and abandoned agricultural soils with different time elapsing since abandonment. (INTF:
iodonitrotetrazolium formazane; BAA: N-a-benzoyl-l-argininamide; PNP: p-nitrophenol. Means are indicated ^ standard deviation; LSD: least signi®cant
differences at P # 0:05†

Elapsed time Dehydrogenase Urease Protease BAA Phosphatase b-Glucosidase

(mg INTF g 21) (mmol NH3 g 21 h 21) (mmol NH3 g 21 h 21) (mmol PNP g 21 h 21) (mmol PNP g 21 h 21)

,10 years 50.0 ^ 6.2 1.38 ^ 0.61 0.63 ^ 0.12 44.8 ^ 9.1 45.1 ^ 12.0
10±20 years 16.2 ^ 5.1 0.63 ^ 0.63 0.52 ^ 0.20 23.7 ^ 8.1 28.2 ^ 11.2
.20 years 16.8 ^ 4.7 0.75 ^ 0.23 0.34 ^ 0.12 30.9 ^ 8.2 21.5 ^ 10.1
Natural soil 61.2 ^ 4.6 1.40 ^ 0.35 1.60 ^ 0.46 127.0 ^ 22.0 105.1 ^ 20.1
LSD 6.1 0.45 0.18 16.2 16.3
 J.A. Pascual et al. / Soil Biology & Biochemistry 32 (2000) 1877±1883 1881

soils as a consequence of the low levels of plant cover

(Brockway et al., 1998) and the low levels of organic matter
(Pascual et al., 1999).
The study of different hydrolase enzyme activities is
important since they indicate the potential of a soil to
carry out speci®c biochemical reactions, and are important
in maintaining soil fertility (Burns, 1982). Urease and
protease (that hydrolyses N-a-benzoyl-l-argininamide) act
in the hydrolysis of organic to inorganic nitrogen, the former
using urea-type substrates and the latter simple peptidic
substrates. Phosphatases catalyse the hydrolysis of organic
phosphorus compounds to phosphates. b-Glucosidase
hydrolyse b-glucosides in soil or in decomposing plant resi-
dues (Hayano and Tubaki, 1985). The overall levels of
hydrolytic enzymes detected in the abandoned soils were
low compared with the soil of the same area which have
not suffered human intervention (Table 3). The existence of
plant cover and the subsequent losses of carbon due to
mineralisation is again shown to be the key to the enzymatic
activities were reduced. Once again, the longest abandoned
soil showed the lowest enzymatic activities.

3.2. Long-term soil remediation after the addition of the

organic fraction of a municipal solid waste

As mentioned above, plant cover is an important factor in

Fig. 3. Total organic carbon (TOC) (A), humic substance carbon (B) and
water-soluble carbon (C) in the amended and unamended soils. (Error bars
denote standard deviation; least signi®cant difference at P # 0:05; TOC ˆ
0:8; humic substance carbon ˆ 140, water-soluble carbon ˆ 30).
soil quality (Brockway et al., 1998), therefore it was
measured 10 years after soil amendment with the organic
fraction of a municipal solid waste (MSW). The percentage
of plant cover on the MSW amended sites increased in the
following order: control plot unamended soil (3% plant
cover) , plot treated with the low dose (25%) ,plot treated
with the high dose (60%). The amendment with organic
matter from MSW had been suf®cient to re-establish a
considerable plant cover that could protect soils against
erosion.
Ten years after the amendment, the TOC content in the
amended soils was higher than in the unamended soil (two
times with the low dose and four times with the high dose)
(Fig. 3A). According to Pascual et al. (1997), approximately
half of the organic matter added as MSW is mineralised
in the ®rst 12 months, thus the increase in organic C
after 10 years was mainly due to the presence of plant
cover and the resulting root exudates and plant residues.
Humic substances and water-soluble compounds also
showed higher values in the amended soil than in the

Table 4
Dehydrogenase and hydrolase enzymes activities of abandoned soil 10 years after organic amendment. (INTF: iodonitrotetrazolium formazane; BAA: N-a-
benzoyl-l-argininamide; PNP: p-nitrophenol. Means are indicated ^ standard deviation; LSD: least signi®cant differences at P # 0:05†

Dehydrogenase Urease Protease BAA Phosphatase b-Glucosidase

(mg INTF g 21) (mmol NH3 g 21 h 21) (mmol NH3 g 21 h 21) (mmol PNP g 21 h 21) (mmol PNP g 21 h 21)

Low dose 13 ^ 3.1 1.60 ^ 0.25 0.51 ^ 0.22 80.0 ^ 20.0 200.0 ^ 20.6
High dose 42 ^ 3.2 3.23 ^ 0.33 1.32 ^ 0.23 180.0 ^ 30.0 370.0 ^ 20.2
Control 8.5 ^ 2.2 1.00 ^ 0.26 0.35 ^ 0.22 40.0 ^ 15.0 25.0 ^ 12.1
LSD 7.1 0.38 0.18 16.6 16.9
1882 J.A. Pascual et al. / Soil Biology & Biochemistry 32 (2000) 1877±1883
Fig. 4. Microbial biomass carbon (A) and basal respiration (B) in the
amended and unamended soils. (Error bars denote standard deviation;
least signi®cant difference at P # 0:05; microbial biomass carbon ˆ 80
and basal respiration ˆ 10.2).
control (Fig. 3B and C). The addition of MSW to soil
abandoned after intensive agricultural use could be a
strategy to preserve and improve long-term soil quality,
due to improvement in the soil structure and the devel-
opment of plant cover.
Soil amendment with MSW gave rise to signi®cantly
…P # 0:05† higher microbial biomass C and basal respira-
tion values than found in the control soil (Fig. 4A). This
improvement could also be attributed to the plant cover
developed as consequence of the initial organic amendment.
In short-term laboratory incubations without vegetation,
microbial biomass C and basal respiration decreased with
length of incubation time, because of the decline in the
easily biodegradable carbon (Pascual et al., 1997). The addi-
tion of MSW under ®eld conditions stimulated the develop-
ment of a plant cover, which acted as a carbon and nutrient
source and which, in principle, maintained the levels of
organic matter, microbial biomass and therefore basal
respiration.
Ten years after the amendment, dehydrogenase activity in
the amended soil was signi®cantly …P # 0:05† higher than
that in the control soil (and signi®cantly higher at the higher
than lower dose, Table 4). This con®rms that the active
microbial biomass is mainly supported by plant root
exudates and residues through out this period.
Amendment with organic matter had a positive effect on
the activity of the different hydrolases studied, probably due
to the higher microbial biomass (Garcia et al., 1994; Pascual
et al., 1998). This suggests that 10 years after the
amendments, the biochemical cycles of N (urease and
protease-BAA activity), P (phosphatase activity) and carbon
(b-glucosidase activity) could have been reactivated, thus
improving the fertility of the amended soil (Ladd, 1985).
The abandonment of Mediterranean soils after intensive
agricultural practices causes a loss of soil quality and the
longer the soils are left, the more degraded they become.
The main reasons for this decline is probably a combination
of the low levels of organic matter, nutrients and microbial
activity. Addition of organic waste after abandoning agri-
cultural use of these soils could be a good strategy to
preserve and improve soil quality for future use. The organic
amendments not only increase the organic matter, but also
lead to an increase in natural vegetation capable of main-
taining high microbial biomass. Knowledge of soil biologi-
cal and biochemical status has helped in the diagnosis of the
capacity for the soil to be regenerated.
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