Translocation of Soil Enzyme Activity by Leachates from Different Agricultural Drainage Systems
Three different agricultural drainage systems located
in the Northeast German lowland i.e., a free drainage of a shallow aquifer,
a submerged deep drainage of a shallow aquifer and a free deep drainage
were studied in order to detect evidence for translocation of enzyme activities
and bacteria in percolating leachates. The activity of fluorescein diacetate
(FDA) hydrolase as a measure of over-all microbial activity in the leachates
was two up to three orders of magnitude lower as compared to the respective
soil substrates. Enzyme activities in the leachates were almost constant
throughout the year, except for a significant increase of FDA activity
and cellulase activity due to a discharge by preferential flow, detected
in the shallow free drainage system. FDA hydrolase was significantly but
weakly correlated with chemical properties (NH4N, DOC). The
population density of culturable bacteria was rather low (50 up to 300
colony-forming units mL-1 drain water) coinciding with a mostly
marginal translocation of soil enzyme activity by leachates from arable
Soil is a dynamic system of inorganic and organic components in
varying proportions, which are the result of interactions between complex
processes such as the weathering of parent materials, the decomposition
and transformation of plant residues and the redistribution of organic
and inorganic materials by water movement. The interactions between hydrological,
organo-mineral and microbiological factors have crucial impact on geochemical
processes and are critical to environmental quality and ecosystem health.
Thus, the role of microorganisms inhabitating the complex, surface-rich
soil environment has long been intensively studied (Foster, 1988; Voroney,
2007), but the complexity of processes and interactions of microorganisms
with mineral surfaces remains hardly understood (Mills and Powelson, 1996;
Nunan et al., 2006). Special emphasis was directed on the survival
and the translocation, or transport of microorganisms in soils. Ever since
molecular genetic approaches became available, microorganisms could be
traced in terrestrial environments at the species level (Thies, 2007),
which is especially important in context with risk assessments of genetically
modified microbial strains (Amarger, 2002; Staley and Brauer, 2006), or
the survival and spread of pathogenic microorganisms in soils (Unc and
It is well described since many decades, that microbial activities are
concentrated in the top soil layers with a s harp decrease of microbial
occurrence and activities with increasing soil depth, as determined by
vertical distribution patterns of soil organic matter contents or root
biomass (Wirth and Wolf, 1992; Salinas-Garcia et al., 1997; Cook
and Kelliher, 2006). Evidence has already accumulated that microbial persistence
and transport in soil is closely associated with physico-chemical properties,
such as the nature of the substratum, the soil solute chemistry and accessibility
of particle surfaces (Lawrence and Hendry, 1996; Fletcher and Murphey,
2001), while passive migration of bacteria or spores in the soil profile
with the water flow seems to be the dominant (passive) transport mechanism
(Huysman and Verstraete, 1993). However, soil microbiology has mostly
focused on the plant root zone and has largely ignored the entirety of
the vadose zone, the unsaturated zone extending from the soil surface
to the groundwater table (Holden and Fierer, 2005). In recent years, however,
attention was drawn on the relevance of protein components in soil leachates
(Schulze, 2005), but hardly any studies are presently available especially
on the translocation of soil organic matter decomposing enzymes with subsurface
Different land use systems, or land reclamation processes such as drainage,
hold the potential to severely impact soil hydraulic properties and thus
soil biochemical processes. Most of the hydromorphic soils in the North-East
German lowland were drained in the last century, providing different water
flow rates and different types of drainage systems i.e., field model systems
with ditches and pipe drainage. The water balance as well as water and
solute transport of selected sites have been previously studied in lysimeter
experiments (Behrendt et al., 2001; Müller et al.,
2001; Schindler et al., 2001). The aim of our study was to detect
evidence for translocation of microbial activity in soils by leachates
in different draining systems on sandy to loamy arable soils. Special
emphasis was laid on the analysis of a temporal sequence of draining events,
on the effect of different drainage systems and on the analysis of correlations
with chemical properties of leachates.
MATERIALS AND METHODS
Study Sites and Soil Properties
The study sites are located in the Pleistocene and Holocene agricultural
landscapes of Brandenburg, north-east Germany i.e., in Seelow (Landkreis
Märkisch-Oderland), Paulinenaue (Landkreis Havelland) and Dedelow
(Landkreis Uckermark). The climate is semi-continental with a mean annual
temperature of 8.5°C and an annual precipitation of 480-550 mm. Typical
soil types of the Pleistocene area are Luvisols and Stagnosols, formed
on sandy to loamy materials. In depressions, Gleysols and Histosols are
frequent, whereas in the lowland of the river Oder, Gleysols on clayey
parent materials are dominant. Prevailing crops on arable sites are cereals
(winter wheat, spring barley), maize and canola, whereas Histosols are
mostly under extensive grassland. For this study, lysimeters as model
drain sites and moreover, several pipe drains in arable field sites were
analysed from February until June 2001, in spring 2002 and again in spring
2006. Thus, different water flow rates were provided by three types of
drainage systems. In Figure 1 drain type A (groundwater
lysimeter at Seelow) represents sites with shallow water table and free
drainage, which are typical for most parts of intensively drained lowland
areas, with both matrix flow and very rapid flow. Figure
1 drain type B (groundwater lysimeter at Paulinenaue)
shows similar conditions, but with submerged drain systems which are typical
for fields with drainage subirrigation systems such as pipe drains, ditches
and weirs. At these less intensively drained sites, discharging rain water
and solutes pass an anaerobic soil zone. Figure 1 drain
type C (moraine field sites at Dedelow) represents typical, most common
pipe drainages in the heterogeneous Pleistocene landscape. Drain outflow
is free but the discharge contains a mix of water from different sources
and ages. Soils of the lysimeters reflect the typical conditions of the
surrounding landscapes. Lysimeters of drain type A contain different soils
ranging from shallow peat soils and sand gleys to clay gleys (Müller
et al., 2001) under arable use. Soils in lysimeters of type B contain
peat soils and humic sand gley soils (Behrendt et al., 2001) under
grassland. Soils of the drain type C are mainly Luvisols of loamy till
rich in calcium (Schindler et al., 2001).
||Schematic draft of the drainage systems at the arable
sites under study. (A) shallow drain, free drainage, (B) deep drain,
submerged drainage and (C) deep drain, free drainage, different travel
Microbiological Analyses of Soils and Leachates
Drain water samples were collected in plastic bottles, stored at 4°C
and analysed within 48 h. Soil samples were sieved (2 mm) and stored at
4°C up to 5 days. Soil enzymes were extracted by suspending soil (1
g) with 25 mL HEPES Puffer (0.5 M, pH 7) and stirring for 60 min at 20°C
(300 rpm). Extracts were finally centrifuged (10 min, 10000 g, 4°C)
and stored on ice. Fluorescein diacetate (FDA) hydrolase as a non-specific
measure of total microbial activity (Schnürer and Rosswall, 1982)
was assayed using a micro-plate approach as described by Wirth (1992).
In detail, 50 μL buffer (HEPES, 0.2 M, pH 7), 50 μL substrate
solution (FDA, 100 μg mL-1) and 100 μL sterile filtered
drain water or soil extract sample were incubated in triplicate at 40°C
up to 4 h. After the addition of ice water (50 μL) to terminate the
reaction, plates were directly assayed spectrophotometrically at 492 nm.
One unit of FDA hydrolase activity was calculated as absorbancex1000xmin-1.
Endo-acting cellulase activity of leachates was assayed colourimetrically
via the detection of reducing sugars with the 3,5-dinitrosalicilic acid
reagent (DNSA, Miller et al., 1960), adapted to 1.5 mL reaction
tubes (Wirth, 1991). In detail, 100 μL HEPES buffer (0.2 M, pH 7),
100 μL carboxymethyl-cellulose substrate solution (CMC, Serva, Germany;
10 mg mL-1) and 200 μL sterile filtered leachate samples
were incubated at 40°C up to 24 h. After the addition of 0.6 mL DNSA
reagent and heating of reaction mixtures in a water bath (15 min, 100°C),
samples were assayed spectrophotometrically at 550 nm. Based on glucose
standards (5 up to 500 μmol glucose monohydrate), one unit of endo-cellulase
activity was defined as equivalent to the release of 1.0 μmol glucose
h-1. The FDA hydrolase and cellulase assay conditions were
optimised in previous experiments with respect to pH, incubation temperature
and substrate concentration.
The total numbers of culturable bacteria in drain waters was determined
as colony forming units (cfu) by a dilution pour-plate method (Ulrich
and Wirth, 1999), using tryptic soy broth, pH 7 (Difco Laboratories, Detroit,
USA; 0.2 g L-1) and bacto-Agar (Difco, 20 g L-1).
Undiluted water samples (1 mL) were inoculated into five replica Petri
dishes and swirled with agar medium at 50°C. Solidified agar plates
were incubated for up to 5 day at 20°C in the dark and counted for
cfu against diffuse light.
Chemical Analyses of Leachates
All drain water samples were collected in plastic bottles, which had
been previously acid washed and soaked in deionized water for 24 h. Samples
were stored at 4°C prior to analysis, which occurred within a few
weeks. All solution analyses, except pH, were measured after filtration
(0.45 μm). The pH was measured with a glass electrode using the WTW
pH Ionmeter pMX 2000. Redoxpotential was determined with a Schott pH-Meter
CG 837. The electrical conductivity was measured with the WTW Ionactivitymeter
LF 95. Ammonium, nitrate and phosphorus were detected colorimetrically
using the Eppendorf EPOS 5060 autoanalyser. Sulphate and chloride were
analysed by Ion chromatography Dionex Dx 320. Calcium was determined by
Flame Atomic Absorption Spectrophotometry Unicam Solar 939.
RESULTS AND DISCUSSION
Chemical Properties of Leachates
The pH values of leachates were generally in the neutral to slightly
alkaline range (pH 7.32 -7.63) and were uniformly distributed throughout
the different drain types (Table 1). The redoxpotential
was low in drain types A and B, which points to anaerobic or microaerophilic
conditions in these soils, whereas the redoxpotential in drain C leachates
was more than twice as high compared to A and B leachates. Samples from
drain type B show the highest electrical conductivity values (2.53 mS
cm-1), caused by high concentrations of chloride and calcium
ions of the corresponding drain waters. The ion concentration of drain
leachates from type A and C were similar and between 2.5 and 3.6 times
lower than drain type B waters. With an average content of 53 mg L-1
Dissolved Organic Carbon (DOC), the drain type B water samples contained
2.8 to 5.2 times higher concentrations than drain waters from A and C
types which is linked to the organic soil type at the lysimeter site.
The ammonium concentrations of the drain waters had a wide concentration
range between 0.01 mg L-1 (type C) and 2.1 mg L-1
(type A) with intermediate contents of 0.98 mg L-1 for type
B. For nitrate, drain type A leachates again had the highest content but
in contrast to ammonium, drain type B leachates had extremely low nitrate
concentrations whereas the type C leachates had medium contents. Overall,
the leached concentrations of DOC, ammonium and nitrate were in a range
commonly reported for soil solutions of arable soils, i.e., 15-50 mg L-1
DOC, 0.2-4 mg L-1 NH4+ and 20-200 mg
L-1 NO3– (Scheffer and Schachtschabel, 2002).
In water samples from type B drains very high chloride concentrations
were detected (mean value = 449 mg L-1), whereas chloride amounts
in type A and type C drain waters were 4.8 and 6.9 times lower. The high
chloride contents of type B drain waters are probably due to the longterm
input of drinking water to the lysimeter soils. In contrast, the sulphate
content of type B leachates were the lowest. This finding is considered
to be a consequence of high adsorption capacities for sulphate in organic
soils. The sulphate concentrations in type A and type C leachates were
in the same range but 2.3 to 3 times higher. The same picture was found
for the phosphorus distribution, i.e., relatively low concentration in
type B water samples and similar amounts in type A and C drain waters,
but 4.4 to 4.9 times higher in concentration. The range of calcium concentrations
in the different drainwater types was not very distinct. The sequence
with increasing concentration was: type C < type A < type B. The
dominant anion in type C water samples was sulphate (SO4/Cl
= 2.2), in type A leachates the portion of each anion is similar (SO4/Cl
= 1.2) and in type B leachates chloride was the dominant anion (SO4/Cl
= 0.1). Soils that exhibit a substantial amount of SO4 adsorption
are considered to be resistant to accelerated cation leaching (Harrison
et al., 1989), but this could not be confirmed by type B leachates,
as these samples showed the highest Ca concentration compared to type
A and C leachates.
||Chemical composition of different drain water types
|a: Mean values, N: No. of samples, SD: Standard
||Extractable fluorescein diacetate hydrolase activities
in different soil substrates and respective activities in leachates
Enzyme Activities in Soils and Respective Leachates
Extracts from soil samples and corresponding drain water samples were
assayed for the activity of FDA hydrolase to provide a measure of over-all
microbial activity, including the activities of esterases and proteases
(Schnürer and Rosswall, 1982). Thus, a soluble extracellular enzyme
complex was analyzed, which is potentially prone to vertical transport.
With respect to the soils and sites under study, FDA hydrolase activities
were highly variable and in a similar range for sand and clay samples,
while activities in peat were lower compared to the mineral substrates
(Table 2). The FDA hydrolase activities in the leachates
were two up to three orders of magnitude lower as compared to the respective
soil substrates. An additional leachate sample derived from a moraine
field site displayed significantly lowest FDA hydrolase activity. Concerning
the different drain types under study, FDA hydrolase activities in leachates
derived from lysimeters were up to threefold higher as compared to a field
drain (Table 3, drain type C). The main factors determining
soil enzyme activity are stocks and availability of substrate, or other
soil properties such as acidity or temperature. Consequently, close associations
between FDA hydrolase activity and organic carbon were described, e.g.,
in forest soil (Wirth, 1992), but in our leachate samples only weak correlations
were found with DOC concentrations (Table 4), probably
due to the high variability and comparatively low activities. Low activities
of FDA hydrolase were also confirmed in corresponding leachate samples
at a later date in May 2006 (data not detailled here). In a general view,
enzymes active in soil are extracellular and highly stabilized to mineral
or organic surfaces (Burns and Dick, 2002), or forming enzyme-substrate
complexes. Consequently, soil enzymes are considered to be protected from
transport by free percolating soil water, but our results give evidence
for considerable translocation of a hydrolytic enyzme. Only few reports
are available on direct evidence of enzyme activity in soil leachates
(e.g., Toor et al., 2003), whereas preferential flow paths are
already recognized as biological hotspots in soil (Bundt et al.,
2001). Further studies are required to underpin these findings, such as
tracing marked enzymes to determine the pattern of transport through intact
lysimeter soil cores.
Enzyme Activities at Different Drainage Events
FDA hydrolase activities of drain water samples were generally low
in late winter or even hardly detectable after prolonged incubation times
(24 h, 40°C, pH 7). Maximum activities were found in
||Fluorescein diacetate hydrolase activities in leachates
from different drainage systems
||Significant correlations between Fluorescein diacetate
hydrolase in the drainwater and chemical properties of drainwater
(All samples, n = 60, α = 0.05)
Parameters not listed are not significant early summer (May), but activities
decreased again in June (Fig. 2a). Similar values of
FDA hydrolase activities in early spring were found in the following year
and confirmed in spring 2006 (data not detailled here). Correspondingly,
the activities of endo-cellulases in leachates were low in spring
and highest in early summer samples (Fig. 2b), but due
to high variability without significant differences. In general, we found
evidence for almost constant enzyme activities in soil leachates throughout
the year, except a significant higher FDA activity and increased cellulase
activity in May 2001, due to a discharge immediately after a heavy rainstorm
of 40 mm resulting in preferential flow. Evidently, the soil pore water
flow conditions are critical for potential transport of enzymes, but more
analyses during a vegetation period would be required to document such
findings more clearly. Preferential flow is a common phenomenon in soils
with pathways for infiltrating water which can be very persistent with
time (Hagedorn and Bundt, 2002). Preferential transport was reported for
anions in clay pan soil, accounting for 35 % of the total transport (Wilkison
and Blevins, 1999). In other cases, pesticides were transported in drain
water by preferential flow (Michaelsen, 1998), or nitrate and pesticide
were transported after periods of excess precipitation (Bosch and Truman,
2002), but to our knowledge no reports are available about translocation
of hydrolytic enzymes with preferential flow. Besides excessive rainfall,
also partly frozen soil needs to be considered to be responsible for rapid
discharge of water (Derby and Knighton, 2001). Moreover, evidence was
reported for the relative contribution of macropores to the movement of
water and transport of organic compounds in dried tire soil (Shipitalo
and Edwards, 1996). Thus, we conclude from present study, that more observance
should be directed on soil enzymes as these biocatalytic compounds can
be subject to vertical translocation with water flow under different soil
Abundance of Bacteria in Leachates
In order to provide more evidence for the generally low enzyme activities
in leachates, bacterial densities were analysed in several drain waters
by using an agar plate technique. As a result, population densities of
50 up to 300 colony-forming units (cfu) of bacteria were determined mL-1
of leachate samples but with a high variability between variants (Fig.
3). In general, the detected densities of bacterial cfu were rather
low, few samples contained >100 cfu mL-1 which is in a range
of cfu concentrations reported for potable waters (Ramalho et al.,
2001). In contrast, densities of bacterial cfu of arable or forest top
soil layers are commonly reported to contain 106 up to 107
cfu g-1 soil (Kauri, 1983; Ulrich and Wirth, 1999). Thus, soil
bacteria are supposed to be rather unlikely transported vertically by
percolating water, or leachates supporting general view that
||FDA hydrolase activities (A) and cellulase activities
(B) of leachates at different drainage periods. FDA: Fluorescein diacetate
hydrolase activity (absorbance x 1000x min-1), CELL: cellulase
activity (1.0 μmol glucose h-1). Site S: Lysimeter
Seelow, site P: Lysimeter Paulinenaue
||Population densities of bacteria in drain water and
surface water samples. cfu: colony-forming units. TSA: Trypticase
Soy Agar. 1-7: lysimeter samples, 8-10: surface samples
microorganisms are sticking to surfaces of particles within the soil
matrix, or are even enmeshed in micro-sites with a steep gradient of microbial
population densities with increasing soil depth in most cases (Mills and
Powelson, 1996). Experimental evidence confirm the view of rather restricted
vertical transport of microbial cells in soil, but depending on soil properties
(Huysman and Verstraete, 1993) or depending on the presence of preferential
flow paths (Bundt et al., 2001). On the other hand, evidence was
provided for vulnerability of soils to leaching of microbes, such as faecal
coliforms, into local surface and groundwater (McLeod et al., 2003).
Further studies would be required to explore translocation of soil bacteria
on species level with special focus on the abundance of unculturable bacteria.
Overall, we thus conclude from present study, that leachates from arable
drainage systems are unlikely to translocate considerable amounts of enzymes,
or bacteria originating from top soil layers, except for preferential
flow events that may cause translocation especially in shallow free drainage
This study was supported by the Federal Ministry of Consumer Protection,
Food and Agriculture (BMVEL) and the Ministry of Agriculture, Environmental
Protection and Area Planning of the State Brandenburg (MLUR). Chemical
analyses were performed by ZALF Central Laboratory.
Dr. Axel Behrendt and Dr. Uwe Schindler supported collection of data
and analysis of some lysimeter samples. Coordination of sampling and data
analysis, field testing of water samples, creating and checking of primary
data files was done by Mrs. Dipl. Ing. (FH) Ute Moritz.
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