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Applied Soil Ecology 30 (2005) 192–202
Effects of N-enriched sewage sludge on soil enzyme activities
Rıdvan Kızılkaya *, Betul Bayraklı
Ondokuz Mayıs University, Faculty of Agriculture, Department of Soil Science, 55139 Samsun, Turkey
Received 16 July 2004; received in revised form 21 February 2005; accepted 21 February 2005
Abstract
Sewage sludge is increasingly used as an organic amendment to soil, especially to soil containing little organic matter.
However, little is known about utility of this organic amendment with N-enriched or adjusted C:N ratios in soil. We studied the
effects of adding of different doses (0, 100, 200 and 300 t ha�1) and C:N ratios (3:1, 6:1 and 9:1) of sewage sludge on enzyme
activities (b-glucosidase, alkaline phosphatase, arylsulphatase and urease) in a clay loam soil at 25 8C and 60% soil water
holding capacity. Nitrogen was added in the form of (NH4)2 SO4 solution to the sludge to reduce the C:N ratio from 9:1 to 6:1 and
3:1. The addition of different doses and C:N ratios of the sludge caused a rapid and significant in the enzymatic activities in soils,
this increase was specially noticeable in soil treated with high doses of the sludge. In general, enzymatic activities in sludge-
amended soils tended to decrease with the incubation time. All activities reached peak values at 30 days incubation and then
gradually decreased up to 90 days of incubation. Sewage sludges also the increased available metal (Cu, Ni, Pb and Zn) contents
in the soils. However, the presence of available soil metals due to the addition of the sludge at all doses and C:N ratios did
negatively affect all enzymatic activities in the soils. This experiment indicated that all doses and C:N ratios of sewage sludge
applied to soil would have harmful effects on enzymatic activity. Some heavy metals found in sewage sludge may negatively
influence soil enzyme activities during the decomposition of the sludge.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Sewage sludge; Soil; C:N ratio; Urease; Alkaline phosphatase; Arylsulphatase; b-Glucosidase activity
1. Introduction
Interest in the disposal of sewage sludges, which
contain a range of valuable nutrients (N, P, Fe, Ca, Mg)
and various other macro- and micro-nutrients essential
for plant growth, on agricultural land has increased
during the last decade (Singh and Pandeya, 1998). One
* Corresponding author. Tel.: +90 362 457 60 20;
fax: +90 362 457 60 34.
E-mail address: [email protected] (R. Kızılkaya).
0929-1393/$ – see front matter # 2005 Elsevier B.V. All rights reserved
doi:10.1016/j.apsoil.2005.02.009
potential use for sewage sludge as a farm fertiliser and/
or soil conditioner is to assist with the growth of arable
land and to help improve and maintain the structure of
the soil by increasing soil aeration and the water
holding capacity of the soil (Pagliai et al., 1981).
Previous studies have demonstrated favorable plant
yield responses to the application of sewage sludge
(King and Morris, 1972). In contrast, the effects of
sewage sludge on biological process in soil have been
questioned by some authors (Knight et al., 1997;
Banerjee et al., 1997).
.
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 193
Most papers concerned with the results of sewage
sludge studies deal with the influence of sewage
sludge on soil biological and enzymatic activity.
Knight et al. (1997) observed a decrease of soil
biological activity such as microbial biomass and
enzyme activities, due to sewage sludge application.
Conversely, Sastre et al. (1996) and Banerjee et al.
(1997) found that the sewage sludge amendment
increased soil microbial activity, soil respiration and
enzyme activities. These differences might be a result
of the heavy metal content of sewage sludge and of the
decomposition rate of the sludge (Tam and Wong,
1990). Sewage sludges are a by-product of sewage
treatment and contain not only nutrients and organic
matter, but also contaminants such as heavy metals
and synthetic organics discharged into the sewers.
Because most heavy metals remain in the soil for a
very long time, any additions should be considered
permanent additions to the total quantity in the soil.
Considerable attention has been focused on the N
content of sewage sludges (Stewart et al., 1975). With
increased cost and shortage of nitrogenous fertilizer,
there is increased emphasis on using sludge for its
nutrient content rather than for disposal. The N content
generally is considered the limiting factor which
determines the application rate of sewage sludges to
agricultural land (Mile and Graveland, 1972).
Increased crop yields have been obtained with the
use of sludges (Hinesly et al., 1972). In some cases
supplemental addition of N further increased yields
(Coker, 1966). Thus low C:N ratio is caused by
additional nitrogen of the sludge. In addition,
numerous studies suggested that different concentra-
tions of nitrogen might predominate in different
sewage sludges (Coker, 1966; Mile and Graveland,
1972; Hinesly et al., 1972). So, the behaviour and
effects of N after incorporation into the soil may not be
similar for different C/N ratios of sludges.
Studies of enzyme activities provide information on
the biochemical processes occurring in soil. There is
growing evidence that soil biological parameters may
be potential and sensitive indicators of soil ecological
stress or restoration. Measurements of several enzy-
matic activities have been used to establish indices soil
biological fertility (Dick and Tabatabai, 1992). In the
present study, soil enzymes representative of main
nutrient cycles (C, N, P, S) were selected. Glucosidases
are widely distributed in nature and their hydrolysis
products as low molecular weight sugars are important
source of energy for soil microorganisms. b-glucosi-
dase catalyzes the hydrolysis of b-D-glucopyranoside
and is one of the three or more enzymes involved in the
saccharification of cellulose (Bandick and Dick, 1999;
Turner et al., 2002).Urease is involved in the hydrolysis
of urea to carbon dioxide and ammonia, which can be
assimilated by microbes and plants. It acts on carbon–
nitrogen (C–N) bonds other than the peptide linkage
(Bremner and Mulvaney, 1978; Karaca et al., 2002).
Phosphatase is an enzyme of great agronomic value
because it hydroles compounds of organic phosphorus
and transforms them into different forms of inorganic
phosphorus, which are assimilable by plants (Amador
et al., 1997). Variations in phosphatase activity apart
from indicating changes in the quantity and quality of a
soil’s phosphorated substrates, are also a good indicator
of its biological state (Pascual et al., 1998, 2002).
Arylsulphatase is the enzyme involved in the hydrolsis
of arylsulphate esters by fission of the oxygen–sulphur
(O–S) bond. This enzyme is believed to be involved in
the mineralisation of ester sulphate in soils (Tabatabai,
1994). Also, it may be an indirect indicator of fungi as
only fungi (not bacteria) contain ester sulphate, the
substrate of arylsulphatase (Bandick and Dick, 1999).
Soil enzymes are biological catalysts of specific
reactions and these reactions depend on a variety of
factors such as pH, temperature and the presence (or
absence) of inhibitors (Burns, 1978). Climate, type of
amendment, cultivation techniques, crop type and
edaphic properties also effect enzyme catalyzed
reactions. Process as related to the degradation of
organic material may be followed through the action of
hydrolases (Pascual et al., 1998). Nannipieri et al.
(1990) pointed out that enzymatic activities are
substrate specific and related to specific reactions.
For this reason it is difficult to obtain an overall picture
of soil status from one enzymatic activity value. The
simultaneous measurement of various enzymes, on the
other hand, seems to be useful to evaluate soil
biochemical activity and related processes (Pascual
et al., 1998).
Indeed, enzyme activities have been shown to be
more sensitive total carbon concentration to soil
management practices. Application of organic wastes,
such as sewage sludge, as a source of organic matter is
a common practice, especially to soils containing little
organic matter, to maintain or improve soil quality and
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202194
Table 1
Total and available metal contents of the soil and the sewage sludge
Total (mg g�1) Available (mg g�1)
Soil Sewage sludge Soil Sewage sludge
Cu 33.7 214.5 1.16 14.4
Ni 46.5 75.8 0.41 1.5
Pb 38.6 180.4 0.36 5.7
Zn 59.1 435.9 0.35 59.4
soil enzymatic activities (Giusquiani et al., 1995).
However, the use of sewage sludge can lead to
problems pertaining to their metal content and their
successive application result in metal accumulation in
soil. In addition, the practice can affect the availability
of metal in soil (Sloam et al., 1997). Moreover, sewage
sludge treatments increase the activity and diversity of
soil microbial populations and their tolerance of
metals (Barkay et al., 1985).
The objective of this study was to determine the
effects of different application rates (0, 100, 200 and
300 t ha�1) and adjusted C/N ratios (3, 6 and 9) of
anaerobically digested sewage sludge on available
metal (Cu, Ni, Pb and Zn) contents and enzyme
activities (b-glucosidase, urease, alkaline phosphatase
and arylsulphatase) in an incubated clay loam soil for
90 days at 25 8C and 60% soil water holding capacity.
2. Materials and methods
2.1. Soil and sludge description
Surface soils (0–20 cm) were taken from the
Experimental Farm of the Agricultural Faculty of
University of Ondokuz Mayıs, Samsun, Turkey. The
soil had been developed from basalt and contained
31.2% clay, 36.2% silt and 32.6% sand. The pH in
water was 7.1, the oxidizable organic matter content
1.98% and the soil C/N ratio was 28:1. The soil was
bulked, all stones, visible roots and fauna removed,
sieved to less than 2 mm and stored at 5 8C until used.
Sewage sludge was obtained from the wastewater
facility set up by the Ankara Wastewater Treatment
Plants, Ankara, Turkey. The sludge was anaerobically
digested with a mixture of primary and waste activated
sludge typically entering the digester. The sewage
sludge was composed of approximately 37% by weight
of oxidable organic matter. The organic fraction
comprised 21% C and 2.3% N while the inorganic
fraction contains 2.3% Fe2O3, 11.5% CaO, 1.3%MgO,
0.2% Na2O and 4.4% Al2O3 by weight. Other elements
of agronomic interest were also analyzed: the sludge
contained 1.3% P and 0.2% K by weight. The sewage
sludge in this experiment was digested and air-dried and
sieved to less than 1 mmand stored in polyethylene bags
at 5 8C until used. The content of the some metal of
interest in the sludge and soil are given in Table 1.
2.2. Soil treatment and incubation
Based on the hypothesis that lowering C/N ratio
enhances C mineralisation was conducted to study the
effect of C:N ratio on enzyme activities in the soil.
Nitrogen was added in the form of (NH4)2 SO4
solution to the sewage sludge to reduce the C:N ratio
from 9:1 to 6:1 and 3:1. The soil samples (250 g air-
dried soil) were placed in 500 ml cylindrical plastic
pots. The sewage sludge was thoroughly mixed with
the soil at a rate equivalent to 0, 100, 200 and
300 t ha�1 on a dried weight basis.
The sewage sludge-amended soils were then
moistened up to 60% soil water holding capacity and
incubated for 90 days at 25 � 0.5 8C. The moisture
contentwasmaintained throughout the experiment. Sub
samples were removed at time intervals of 7, 15, 30, 45,
60 and 90 days to determine the changes in enzyme
activities.
2.3. Experimental design
Soil without sewage sludge addition was used as a
control. A randomized complete plot design with three
replicates per treatment and soil was used. The
experiment was performed with the following 10
treatments:
(1) c
ontrol(2) +
100 t ha�1 sewage sludge (C/N ratio 3:1)(3) +
200 t ha�1 sewage sludge (C/N ratio 3:1)(4) +
300 t ha�1 sewage sludge (C/N ratio 3:1)(5) +
100 t ha�1 sewage sludge (C/N ratio 6:1)(6) +
200 t ha�1 sewage sludge (C/N ratio 6:1)(7) +
300 t ha�1 sewage sludge (C/N ratio 9:1)(8) +
100 t ha�1 sewage sludge (C/N ratio 6:1)(9) +
200 t ha�1 sewage sludge (C/N ratio 6:1)(10) +
300 t ha�1 sewage sludge (C/N ratio 9:1)R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 195
2.4. Measurement of soil enzymatic activities
b-Glucosidase (EC 3.2.1.21) activity (GA) was
measured according to Eivazi and Tabatabai (1988).
0.25 ml toluene, 4 ml Tris (hydroximethyl) amino-
methane buffer (pH 12) and 1 ml of 0.05 M p-
nitrophenyl b-D-glucopyranoside solution were added
to the 1 g sample and the samples were incubated for
1 h at 37 8C. The formation of p-nitrophenol was
determined spectrophotometrically 410 nm and
results were expressed as mg p-nitrophenol g�1 dry
sample.
Urease (EC 3.5.1.5) activity (UA) was measured by
the method of Hoffmann and Teicher (1961). 0.25 ml
toluene, 0.75 ml citrate buffer (pH 6.7) and 1 ml of
10% urea substrate solution were added to the 1 g
sample and the samples were incubated for 3 h at
37 8C. The formation of ammonium was determined
spectrophotometrically at 578 nm and results were
expressed as mg N g�1 dry sample.
Alkaline phosphatase (EC 3.1.3.1) activity (APA)
was determined according to Tabatabai and Bremner
(1969). 0.25 ml toluene, 4 ml phosphate buffer (pH
8.0) and 1 ml of 0.115 M p-nitrophenyl phosphate
(disodium salt hexahydrate) solution were added
to the 1 g sample and the samples were incubated for
1 h at 37 8C. The formation of p-nitrophenol was
determined spectrophotometrically at 410 nm and
results were expressed as mg p-nitrophenol g�1 dry
sample.
Arylsulphatase (EC 3.1.6.1) activity (ASA) was
measured according to Tabatabai and Bremner (1970).
0.25 ml toluene, 4 ml acetate buffer (pH 5.5) and 1 ml
of 0.115 M p-nitrophenyl sulphate (potassium salt)
solution were added to the 1 g sample and the samples
were incubated for 1 h at 37 8C. The formation of
p-nitrophenol was determined spectrophotometrically
410 nm and results were expressed as mg p-
nitrophenol g�1 dry sample.
All determinations of enzymatic activities were
performed in triplicate, and all values reported are
averages of the three determinations expressed on an
oven-dried soil basis (105 8C).
2.5. Measurement of soil metal contents
Available metals in soils were determined by
extraction with diethylene triamine penta acetic acid
(DTPA) solution (0.005 M DTPA + 0.01 M
CaCI2 + 0.1 M TEA buffered at pH 7.3) and analyzed
by atomic absorption spectrophotometer (Lindsay and
Norvell, 1978).
2.6. Statistical analysis
Means and standard deviation of triplicates were
determined and all the figures presented include
standard errors of the data. Analysis of variance
(three-way ANOVA) was carried out using three
factors randomized complete plot design (C/N
ratios � doses of sewage sludge � incubation period).
The means were compared using least significant
difference (LSD) test, with a significance level of
P < 0.01. All statistical calculations were performed
using MSTAT and SPSS 11.0 computer programs.
3. Results and discussion
3.1. Metal contents of the sewage sludge
The total Cu, Ni, Pb and Zn concentrations in
the sewage sludge were 214.5, 75.8, 180.4 and
435.9 mg g�1, respectively. The concentrations were
similar to those reported by Arcak et al. (2000),
Karaca and Haktanır (2000) and Kızılkaya (2004) for
same sewage sludge treatment works. However, the
concentrations were relatively low when compared to
sewage sludge originating from industrialized cities in
Europe (Kelly et al., 1999). The concentrations of Cu,
Ni, Pb and Zn in the soil sampled are shown in Table 1.
3.2. Available metal concentrations
The changes of metal (Cu, Ni, Pb and Zn)
availability in sewage sludge-amended soils during
the incubation are shown in Fig. 1.
Different C/N ratios of sewage sludge application
significantly affected the levels and distribution of
available (DTPA-extractable) Cu, Ni, Pb and Zn in the
soil, when compared with the control treatment. Fig. 1
shows that the Cu, Ni, Pb and Zn concentrations of the
low C/N ratio and high doses of sewage sludge
application were higher than those of other treatments
at all sampling times. In general sewage sludge
treatments with initial high C/N ratios had low metal
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202196
Fig. 1. Changes in available metals (Cu, Ni, Pb and Zn) of soil amended with sewage sludge during the incubation period. Vertical bars indicate
standard error of mean of three replicates at 95% confidence level.
concentrations at all sludge doses. This situation might
be related availability of metals such as initial C/N
ratios, doses of sewage sludge and chemical condi-
tions (organic matter, pH, etc.) in soil. Organic wastes
as sewage sludge and their C/N ratios are the most
important factors that control the availability of heavy
metals in soil (Doelman and Haanstra, 1984). The
concentration of Cu, Ni, Pb and Zn to the soil can be
attributed to the high affinity of the metals to organic
matter (Tyler and McBride, 1982; McGrath and Lane,
1989). The study, although based on one soil type, was
in agreement with the results by McGrath et al. (1994)
and Nyamangara and Mzezewa (1999) who reported
that generally metal concentrations relatively
increased in sewage sludge treated soil compared to
the non-treated soil.
3.3. Enzyme activities
Considerable variations in all enzyme activities,
GA, APA, ASA and UA were found for the different
doses and C:N ratios of sewage sludge at different
sampling times. Statistically significant variations
were found in all enzyme activities at various
application rates and C/N ratios of the sewage sludge.
Enzyme activities were also affected by incubation
period. The analysis of variance of the results obtained
in our experiment on the periodic sampling times with
sewage sludge showed that all factors (sewage sludge
doses, C/N ratios and incubation periods) significantly
influenced all enzyme activities (Table 2). After
sewage sludge addition a rapid and significant
increase in enzymatic activities was observed in
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 197
Table 2
Results of ANOVA
Variables GA APA ASA UA
F-value LSDa = 0.01 F-value LSDa = 0.01 F-value LSDa = 0.01 F-value LSDa = 0.01
C:N ratio (CN) 58.37*** 1.608 22.04*** 2.151 0.61ns 1.314 33.56*** 1.027
Sewage sludge doses (DS) 453.56*** 1.856 90.52*** 2.484 4.21** 1.518 445.41*** 1.186
Incubation period (IN) 221.43*** 2.274 315.57*** 3.042 172.00*** 1.453 583.37*** 1.453
CN � DS 8.56*** 3.125 8.96*** 3.255 0.22ns 2.629 8.92*** 2.054
CN � IN 2.23* 3.938 3.65** 5.270 4.66*** 3.220 14.55*** 2.516
DS � IN 53.41*** 4.547 53.06*** 6.085 52.11*** 3.718 150.48*** 2.905
CN � DS � IN 0.85ns 7.876 1.54ns 10.539 0.75ns 6.439 3.26*** 5.032
ns: Not significant.* P < 0.05.** P < 0.01.*** P < 0.01.
sludge-amended soils followed by a progressive
decrease in the GA, UA, APA and ASA in soils
amended with the sludge. At the end of the incubation,
the GA, UA, APA and ASA measured in treated soils
were statistically different from those measured in the
control soils.
Among the C:N ratios and application doses of
sludge, soils amended with high C:N ratio and high
doses of sludge presented the highest values of GA
(Fig. 2). The GA is closely involved in the C cycle and
plays an important role in the hydrolytic processes that
take place during the organic matter breakdown
(Bandick and Dick, 1999; Turner et al., 2002). The GA
in soils treated with sludges can be attributed to high
levels of GA in the material itself and the stimulation
of microbial activity (Martens et al., 1992).
Alkaline phosphatase activity showed a similar
trend to that of ASA in both soils: an increase in the
first 30 days of incubation followed by a pronounced
decrease in activities (Figs. 3 and 4). The APA and
ASA plays an essential role in the mineralisation of
organic P and organic S in soil, respectively (Speir and
Ross, 1978). The APA and ASA of low C:N ratio and
high doses of sludge treatments were higher than those
of other treatments. Thus indicated that there were
higher amounts of readily available substrates in the
substrates in the sewage sludge initially. Organic
matter in sludge usually contains high amounts of
APA and ASA substrates (Garcıa et al., 1993).
Urease activity (UA) exhibited the same behaviour
as those of the APA and ASA. However, the UA
increased rapidly and reached a maximum activity
after 15 days for all treatments; then the UA decreased
significantly until the end of the incubation period
(Fig. 5). The UA is involved in the hydrolysis C–N
bonds of some amides and urea (Bremner and
Mulvaney, 1978). The highest values of UA were
observed in soil amended with low C:N ratio and high
doses of sludge, probably due to the UA is a
constitutive-intracellular enzyme and consequently
it increases or decreases proportional to microbial
biomass (Nannipieri et al., 1979).
Nitrogen can have different effects on decomposi-
tion rates depending on the stage of decomposition.
The initial stage of litter decomposition will often be
N limited, and consequently, addition of N will
enhance the degradation rate. Several studies (Cotrufo
et al., 1995; Recous et al., 1995) have demonstrated
that low C/N ratio is an indicative of a high
decomposition rate.
In general, GA, UA, APA and ASA in sludge-
amended soils decreased dramatically of 30 days of
incubation at all C:N ratios and doses of sludge.
Taking into account the evolution of the enzyme
activities during the incubation period, two different
stages can be distinguished: a first stage lasting 30
days during which a great activation of the enzymes as
a result of adding the different doses and C:N ratios of
sewage sludge was recorded, and a second stage
lasting until the end of the incubation in which a major
decrease in the enzyme activities in soils treated with
the sludge was recorded. The first stage depends on
microbial growth and available substrate for enzymes
(Garcıa et al., 1993) stimulated by addition of sewage
sludge to soil. Thus, increased microbial activity may
have synthesized enzymes in soil (Bremner and
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202198
Fig. 2. Changes in b-glucosidase activity (GA) of soil amended
with sewage sludge during the incubation period. Vertical bars
indicate standard error of mean of three replicates at 95% confidence
level.
ig. 3. Changes in alkaline phosphatase activity (APA) of soil
mended with sewage sludge during the incubation period. Vertical
ars indicate standard error of mean of three replicates at 95%
onfidence level.
F
a
b
c
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 199
Fig. 4. Changes in arylsulphatase activity (ASA) of soil amended
with sewage sludge during the incubation period. Vertical bars
indicate standard error of mean of three replicates at 95% confidence
level.
Fig. 5. Changes in urease activity (UA) of soil amended with
sewage sludge during the incubation period. Vertical bars indicate
standard error of mean of three replicates at 95% confidence
level.
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202200
Mulvaney, 1978; Bandick and Dick, 1999). The
second stage depends on contents and availability of
sludge borne metals.
In general, enzymatic activity diminished with
increasing available concentration of metals (Tyler,
1974; Baath, 1989; Kızılkaya et al., 2004). In this
study, sewage sludge application significantly affected
the levels of available Cu, Ni, Pb and Zn concentra-
tions in soil, when compared with control (Fig. 1).
Similarly, several studies demonstrated that enzyme
activities decreased with increasing metal concentra-
tions using sewage sludge (Madojon et al., 2001;
Karaca et al., 2002; Moreno et al., 2003).
Nannipieri (1994) and Kandeler et al. (2000)
reported that the effect of sludge borne metals on
enzyme activities is very complex and is due to: (1) the
metal reacting with sulphydral groups of enzymes
causing inhibition or inactivation of the enzymatic
activity; (2) metals indirectly affecting soil enzymatic
activities by altering the microbial community which
synthesizes enzymes; or (3) a combination of these
factors. Assays of soil enzyme activity are important
in estimating the effects of metal pollution on the soil
environment. It is difficult, however, to establish
whether low enzymatic activity occur because of
metal inhibition, or from low concentrations of the
enzyme resulting from impeded microbial growth and
enzyme synthesis (Tyler, 1974). The metal suscept-
ibility of enzyme activities was reported by Kandeler
et al. (1996), Kuperman and Carreiro (1997) and
Kunito et al. (2001). The inhibition of enzyme
activities by metals in soils is principally attributed
to an indirect effect (suppression of microbial
population and cellular activities) and/or a direct
effect (inactivation of extracellular enzymes due to
binding with metals). Also, Geiger et al. (1998a, b)
reported the direct inactivation of an extracellular
enzyme by metals due to their binding to some amino
acids in the enzyme. Because GA, APA and ASA are
present extracellularily in the soils (Bremner and
Mulvaney, 1978; Bandick and Dick, 1999) the
suppression might be attributable to the fact that
metal suppressed the number of microorganisms
producing the enzymes and the microbial activity
and that the extracellular enzymes were directly
inactivated by metals. The results described here on
the suppression of GA, UA, APA and ASA would
indicate the disruption of soil function by the metal
contamination in the soils. Nevertheless, soil enzy-
matic activity is believed to be a sensitive indicator of
the effect of environmental factors on microbial
functions (Dick, 1992). Thus, because of their role in
nutrient cycling, enzymes like GA, UA, APA and ASA
are suggested to be good indicators of potentially
beneficial or harmful effects on the soil.
The negative effect of metals on enzymatic
activities might have been masked in our study by
the positive effect of the sewage sludge (Moreno et al.,
1999). In addition, a metal fraction might be adsorbed
on the organic colloids added with the sludge. The
rapid decomposition of organic matter which occurs
after the application of composts to soil, increases the
proportion of available metals as a result of miner-
alisation of organically complexed (OM–) metals
(Dudley et al., 1986). The OM– heavy metal fractions,
which are readily available for plant uptake, occur in
organic matter and soil solutions. This would prevent
the heavy metal from interacting directly with the
active sites of enzyme, thus affecting the enzyme’s
activity (Doelman and Haanstra, 1984).
4. Conclusions
At present, many risk assessments and regulations
for the application of sewage sludge in many countries
are based on total and available metal concentrations
in soil (McGrath et al., 1994). In this study,
considerable effort has been devoted to estimating
the fate of enzymatic activity in the sludge-amended
soils. A good N balance is critical factor in any land
application program. High amounts of N in the initial
sludge application or N fertilization in the sludge-
amended soil can result in enzyme inhibition.
However, our present study suggests that measure-
ments of only available metal (Cu, Ni, Pb and Zn)
contents and hydrolytic enzyme activities (GA, UA,
APA and ASA) cannot provide a presence prediction
of toxicity other extracellular and intracellular
enzymes in soils amended with sewage sludge,
indicating that their scientific validity is questioned.
Hence, further studies are needed to explore the
relevancy in using enzyme activity to reflect different
types of sewage sludge and soil. This would not only
overcome problems of enzyme inhibition but also
would reduce a major area of public concern such as
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202 201
nitrate leaching, heavy metal and pathogen contam-
ination, plant uptake of sludge borne metals and soil
fertility and health.
Acknowledgements
The authors thank Dr. Cafer Turkmen of Ankara
University for his assistance with sewage sludge
collection. This work was supported by the OMU
Center of Scientific Research (project number Z-385).
References
Amador, J.A., Glucksman, A.M., Lyons, J.B., Gorres, J.H., 1997.
Spatial distribution of soil phosphatase activity within a riparian
forest. Soil Sci. 162, 808–825.
Arcak, S., Karaca, A., Haktanır, K., 2000. Investigations on
sewage sludges: chemical composition and effects on some
chemical properties of soil. In: Proceedings of International
Symposium on Desertification (ISD), Konya, Turkey, 13–17
June.
Baath, E., 1989. Effects of heavy metals in soil on microbial
processes and populations (a review). Water Air Soil Pollut.
47, 335–379.
Bandick, A.K., Dick, R.P., 1999. Field management effects on soil
enzyme activities. Soil Biol. Biochem. 31, 1471–1479.
Banerjee, M.R., Burton, D.L., Depoe, S., 1997. Impact of sewage
sludge application on soil biological characteristics. Agric.
Ecosyst. Environ. 66, 241–249.
Barkay, T., Tripp, S.C., Olson, B.H., 1985. Effect of metal rich
sewage sludge application on the bacterial communities of
grassland. Appl. Environ. Microb. 49, 333–337.
Bremner, J.M., Mulvaney, R.L., 1978. Urease activity in soils. In:
Burns, R.G. (Ed.), Soil Enzymes. Academic Press, New York,
pp. 149–196.
Burns, R.G., 1978. Enzyme activity in soil. Some theoretical and
practical considerations. In: Burns, R.G. (Ed.), Soil Enzymes.
Academic Press, New York, pp. 295–339.
Coker, E.G., 1966. The value of liquid digested sewage sludge II.
Experiments on rye grass in south-east England, comparing
sludgewith fertilizer supplying equivalent nitrogen, phosphorus,
potassium, and water. J. Agric. Sci. 67, 99–103.
Cotrufo, M.F., Ineson, P., Roberts, J.D., 1995. Decomposition of
birch leaf litters with varying C-to-N ratios. Soil Biol. Biochem.
27, 1219–1221.
Dick, R.P., 1992. A review: long-term effects of agricultural systems
on soil biochemical and microbiological parameters. Agric.
Ecosyst. Environ. 40, 25–36.
Dick, W.A., Tabatabai, M.A., 1992. Potential uses of soil enzymes.
In: Meeting, F.B. (Ed.), Soil Microbial Ecology: Applications in
Agricultural and Environmental Management. Marcel Dekker,
New York, pp. 95–127.
Doelman, P., Haanstra, L., 1984. Short-term and long-term effects of
cadmium, chromium, copper, nickel, lead and zinc on soil
microbial respiration in relation to abiotic soil factors. Plant
Soil 79, 317–327.
Dudley, L.M., McNeal, B.L., Baham, J.E., 1986. Time-dependent
changes in soluble organics, copper, nickel, and zinc from
sludge-amended soils. J. Environ. Qual. 15, 188–192.
Eivazi, F., Tabatabai, M.A., 1988. Glucosidases and galactosidases
in soil. Soil Biol. Biochem. 20, 601–606.
Garcıa, C., Hernandez, T., Costa, C., Ceccanti, B., Masciandaro, G.,
Ciardi, C., 1993. A study of biochemical parameters of com-
posted and freshmunicipal wastes. Bioresource Technol. 44, 17–
23.
Geiger, G., Livingston, M.P., Funk, F., Schulin, R., 1998a. b-
Glucosidase activity in the presence of copper and geothite.
Eur. J. Soil Sci. 49, 17–23.
Geiger, G., Brandl, H., Furrer, G., Schulin, R., 1998b. The effect of
copper on the activity of cellulase and b-glucosidase in the
presence of montmorillonite or Al-montmorillonite. Soil Biol.
Biochem. 30, 1537–1544.
Giusquiani, P.L., Pagliai, M., Gigliotti, G., Businelli, D., Benetti, A.,
1995. Urban waste compost: effects of physical, chemical and
biochemical soil properties. J. Environ. Qual. 24, 175–182.
Hinesly, T.D., Jones, R.L., Ziegler, E.L., 1972. Effects on corn
applications of heated anaerobically digested sludge. Compost
Sci. 13, 26–30.
Hoffmann, G.G., Teicher, K., 1961. Ein Kolorimetrisches Verfahren
zur Bestimmung der Urease Aktivitat in Boden. Z. Pflanzener-
nahr. Bodenk. 91, 55–63.
Kandeler, E., Kampichler, C., Horak, O., 1996. Influence of heavy
metals on the functional diversity of soil microbial communities.
Biol. Fertil. Soils 23, 299–306.
Kandeler, E., Tscherko, D., Bruce, K.D., Stemmer, M., Hobbs, P.J.,
Bardgett, R.D., Amelung, W., 2000. Structure and function of
the soil microbial community in microhabitats of a heavy metal
polluted soil. Biol. Fertil. Soils 32, 390–400.
Karaca, A., Haktanır, K., 2000. Arıtma camurlarının toprakta alın-
abilir kursun ve dehidrogenaz enzim aktivitesi uzerine etkileri.
A. U. Z. F. Tarım Bilimleri Dergisi 6 (3), 13–19.
Karaca, A., Naseby, D.C., Lynch, J.M., 2002. Effect of cadmium
contamination with sewage sludge and phosphate fertiliser
amendments on soil enzyme activities, microbial structure
and available cadmium. Biol. Fertil. Soils 35, 428–434.
Kelly, J.J., Max Haggblomb, M., Tate III, R.L., 1999. Effects of the
land application of sewage sludge on soil heavy metal concen-
trations and soil microbial communities. Soil Biol. Biochem. 31,
1467–1470.
Kızılkaya, R., 2004. Cu and Zn accumulation in earthworm L.
terrestris L. in sewage sludge-amended soil and fractions of
Cu and Zn in casts and surrounding soil. Ecol. Eng. 22, 141–151.
Kızılkaya, R., Askın, T., Bayraklı, B., Saglam, M., 2004. Micro-
biological characteristics of soils contaminated with heavy
metals. Eur. J. Soil Biol. 40, 95–102.
King, L.D., Morris, H.D., 1972. Land disposal of liquid
sewage sludge. 2. The effects on soil pH, manganese, zinc,
and growth and chemical composition of rye. J. Environ. Qual. 1,
325–329.
R. Kızılkaya, B. Bayraklı / Applied Soil Ecology 30 (2005) 192–202202
Knight, B.P., McGrath, M.J., Doran, J.W., Cline, R.G., Harris, R.F.,
Schuman, G.E., 1997. Biomass carbon measurements and sub-
strate utilization patterns of microbial populations from soils
amended with cadmium, copper, or zinc. Appl. Environ. Microb.
63, 39–43.
Kunito, T., Saeki, K., Goto, S., Hayashi, H., Oyaizu, H., Matsumoto,
S., 2001. Copper and zinc fractions affecting microorganisms in
long-term sludge-amended soils. Bioresource Technol. 79, 135–
146.
Kuperman, M.M., Carreiro, R.G., 1997. Soil heavy metal concen-
trations, microbial biomass and enzyme activities in a contami-
nated grassland ecosystem. Soil Biol. Biochem. 29, 179–190.
Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil
test for zinc, iron, manganese and copper. Soil Sci. Soc. Am. J.
42, 421–428.
Madojon, E., Burgos, P., Lopez, R., Cabrera, F., 2001. Soil enzy-
matic response to addition of heavy metals with organic resi-
dues. Biol. Fertil. Soils 34, 144–150.
Martens, D.A., Johanson, J.B., Frankenberger, W.T., 1992. Produc-
tion and persistence of soil enzyme with repeated addition of
organic residues. Soil Sci. 153, 53–61.
McGrath, S.P., Lane, P.W., 1989. An explanation for the apparent
losses of metals in long-term field experiment with sewage
sludge. Environ. Pollut. 60, 235–256.
McGrath, S.P., Chang, A.C., Page, A.L., Witter, E., 1994. Land
application of sewage sludge: scientific perspectives of heavy
metal loading limits in Europe and the US. Environ. Rev. 2, 108–
118.
Mile, R.A., Graveland, D.N., 1972. Sewage sludge as a fertilizer.
Can. J. Soil Sci. 52, 270–273.
Moreno, J.L., Hernandez, T., Garcıa, C., 1999. Effects of a cadmium
contaminated sewage sludge compost on dynamics of organic
matter and microbial activity in an arid soil. Biol. Fertil. Soils 28,
230–237.
Moreno, J.L., Garcıa, C., Hernandez, T., 2003. Toxic effect of
cadmium and nickel on soil enzymes and the influence of adding
sewage sludge. Eur. J. Soil Sci. 54, 377–386.
Nannipieri, P., 1994. The potential use of soil enzymes as
indicators of productivity, sustainability and pollution. In:
Pankhurst, C.E., Double, B.M., Gupta, V.V.S.R., Grace, P.R.
(Eds.), Soil Biota. Management in Sustainable Farming Sys-
tems. CSIRO, East Melbourne, pp. 238–244.
Nannipieri, P., Greco, S., Ceccanti, B., 1990. Ecological significance
of the biological activity in soil. In: Bollag, J.M., Stotzky, G.
(Eds.), Soil Biochemistry, vol. 6. Marcel Dekker, New York, pp.
293–355.
Nannipieri, P., Pedrazzini, F., Arcara, P.G., Piovanelli, C., 1979.
Changes in amino acids, enzyme activities, and biomass during
soil microbial growth. Soil Sci. 127, 26–34.
Nyamangara, J., Mzezewa, J., 1999. The effect of long-term sewage
sludge application on Zn, Cu, Ni and Pb levels in a clay loam soil
under pasture grass in Zimbabwe. Agric. Ecosys. Environ. 73,
199–204.
Pagliai, M., Guidi, G., La Marca, M., Giachetti, M., Lucamante, G.,
1981. Effects of sewage sludges and composts on soil porosity
and aggregation. J. Environ. Qual. 10, 556–561.
Pascual, J.A., Hernandez, T., Garcia, C., Ayuso, M., 1998. Enzy-
matic activities in an arid soil amended with urban organic
wastes: laboratory experiment. Bioresource Technol. 64, 131–
138.
Pascual, J.A., Moreno, J.L., Hernandez, T., Garcıa, C., 2002.
Persistence of immobilised and total rease and phosphatase
activities in a soil amended with organic wastes. Bioresource
Technol. 82, 73–78.
Recous, S., Robin, D., Darwis, D., Mary, B., 1995. Soil inorganic N
availability: effect on maize residue decomposition. Soil Biol.
Biochem. 27, 1529–1538.
Sastre, I., Vicente, M.A., Lobo, M.C., 1996. Influence of the
application of sewage sludges on soil microbial activity. Bior-
esource Technol. 57, 19–23.
Singh, A.K., Pandeya, S.B., 1998. Modelling uptake cadmium
by plants in sludge-treated soils. Bioresource Technol. 66,
51–58.
Sloam, J.J., Doedg, R.H., Dolan, M.S., Linden, D.R., 1997. Long-
term effects of biosolid applications on heavy metal. Bioavail-
ability in agriculturaal soils. J. Environ. Qual. 26, 966–974.
Speir, T.W., Ross, D.J., 1978. Soil phosphatase and sulphatase. In:
Burns, R.G. (Ed.), Soil Enzymes. Academic Press, New York,
pp. 197–250.
Stewart, N.E., Beauchamp, E.G., Corke, C.T., Webber, L.R., 1975.
Nitrate nitrogen distribution in corn land following applications
of digested sewage sludge. Can. J. Soil Sci. 55, 287–294.
Tabatabai, M.A., 1994. Soil enzymes. In: Mickelson, S.H., Bighan,
J.M. (Eds.), Methods of Soil Analysis. Part 2. Microbiological
and Biochemical Properties. Soil Science Society of America,
Madison, pp. 775–826.
Tabatabai, M.A., Bremner, J.M., 1969. Use of p-nitrophenyl phos-
phate for assay of soil phosphatase activity. Soil Biol. Biochem.
1, 301–307.
Tabatabai, M.A., Bremner, J.M., 1970. Arylsulphatase activity of
soils. Soil Sci. Soc. Am. Proc. 34, 225–229.
Tam, N.F.Y., Wong, Y.S., 1990. Respiration studies on the decom-
position of organic waste-amended colliery spoil. Agric. Eco-
syst. Environ. 32, 25–38.
Turner, B.L., Hopkins, D.W., Haygarth, P.M., Ostle, N., 2002. b-
Glucosidase activity in pasture soils. Appl. Soil Ecol. 20, 157–
162.
Tyler, G., 1974. Heavy metal pollution and soil enzymatic activity.
Plant Soil 41, 303–311.
Tyler, L.D., McBride, M.B., 1982. Mobility and extractability of
cadmium, copper, nickel, and mineral soil columns. Soil Sci.
134, 198–225.
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