J00394
Soil Biology & Biochemistry 41 (2009) 243–250
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Changes in nitrification and bacterial community structure upon crossinoculation of Scots pine forest soils with different initial nitrification rates
Rully A. Nugroho a, Wilfred F.M. Ro¨ling b, *, Nico M. van Straalen a, Herman A. Verhoef a
a
b
Institute of Ecological Science, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2008
Received in revised form
13 October 2008
Accepted 17 October 2008
Available online 12 November 2008
Nitrification occurs slowly in many acid Scots pine forest soils. We examined if bacterial community
structure and interactions between members of the bacterial community in these forest soils prohibit
growth of ammonia-oxidising microorganisms and their nitrifying activity. Native and gamma-irradiated
Scots pine forest soils known to have low net nitrification rates were augmented with fresh soils or soil
slurries from nitrifying Scots pine forest soil, and vice versa. Augmentation of native non-nitrifying soils
with nitrifying soils induced net nitrification, although no significant changes in bacterial community
structure, as measured by 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE), were
observed. In sterilised soils, the inoculum, i.e. native nitrifying soil or non-nitrifying soil, determined the
occurrence of net nitrification and bacterial community structure, and not the origin of the sterilised
soils. Our results demonstrate that low net nitrification rates in acid Scots pine forest soils cannot be
(solely) explained by unfavourable abiotic soil conditions, but that still uncaptured biotic factors
contribute to suppression of nitrification.
Ó 2008 Elsevier Ltd. All rights reserved.
keywords:
Cross-inoculation
Bioaugmentation
Net nitrification
Bacterial communities
Scots pine
1. Introduction
Nitrification tests using soil incubations have revealed that
nitrification occurred readily in some acid pine forest soils, but
slowly in others (Bottomley et al., 2004; Nugroho et al., 2005,
2007). Differences in the occurrence and nitrification rate can in
part be explained by abiotic factors. Acid forest soils with low net
nitrification rates are correlated with high C/N ratios (or low total
N) and low atmospheric N depositions (Ba¨ckman et al., 2003;
Compton et al., 2004; Nugroho et al., 2005, 2007; Persson and
Wire´n, 1995; Tolli and King, 2005). Low net nitrification rates in
acid forest soils cannot be explained solely by soil pH, since soil pH
values did not differ significantly between soils with low and high
net nitrification rates (Nugroho et al., 2005). N availability also does
not constrain net nitrification in these soils as large amounts of
NHþ
4 -N are produced during incubation of these soils in the laboratory (Booth et al., 2005; Nugroho et al., 2005; Persson and Wire´n,
1995). As in some acid soils with increased NHþ
4 -N concentrations
net nitrification does not occur (Nugroho et al., 2007), other
suppressive factors need to be considered.
Differences in nitrification could also be driven by biotic factors.
Nitrification rates might be linked to the composition of the soil
* Corresponding author. Tel.: þ31 20 598 7192; fax: þ31 20 598 7229.
E-mail address: wilfred.roling@falw.vu.nl (W.F.M. Ro¨ling).
0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2008.10.020
bacterial community (Balser and Firestone, 2005; de Boer and Kester, 1996; de Boer et al., 1996; Wheatley et al., 2003). Community
members can have specific positive or negative effects on the growth
and activity of the nitrifying bacteria. Chitinolytic soil bacteria can
produce antibiotics against nitrifying bacteria (de Boer et al., 1996).
Ammonia-oxidising bacteria (AOB) are poor competitors for NHþ
4
relative to ammonia-assimilating heterotrophs when ammonium is
limiting (van Niel et al., 1993; Verhagen and Laanbroek, 1991; Verhagen et al., 1992, 1995). Wheatley et al. (2003), using a nested PCR
with the not very specific CTO primer set, also indicated that nitrification rates might be linked to the composition of the soil bacterial
community, rather than to the AOB community itself. They
compared the structure of the bacterial community within and
between three arable fields differing in potential nitrification rates
but broadly similar in basic characteristics (soil pH, total C and total
N contents). The bacterial community structure in each field differed
significantly. In contrast, molecular analyses specific to AOB suggested that the populations in all three fields were similar in types
and did not vary with time (Wheatley et al., 2003).
The relationships between overall bacterial community structure and nitrification rates in acid Scots pine forest soils have not
been studied thoroughly. Previous studies (Laverman et al., 2000;
Nugroho et al., 2005), applying specific inhibitors of autotrophic
nitrification, have revealed that heterotrophic nitrification does not
play a significant role in such soils. Our aim was to examine if
general bacterial community structure and interactions between
244
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
members of the bacterial community in acid Scots pine forest soils
with low nitrification rate prohibit growth of ammonia oxidizers
and their nitrifying activity. If so, we may expect that when native
soils (showing either low or high nitrification rates) are inoculated,
or augmented, with a small quantity of forest soil with contrasting
nitrification activity, no significant changes in nitrification will
occur. However, we would expect that when the soil microbial
community is destroyed by sterilisation, the nitrifying potential of
the inoculum will be able to establish itself in the sterilised soil,
unless abiotic conditions in the sterilised soil prohibit this. To test
these hypotheses, native and gamma-irradiated Schoorl (The
Netherlands) soils, with low nitrification rates (Nugroho et al.,
2005, 2007), were augmented with fresh soils or soil slurries from
nitrifying Wekerom soil (The Netherlands) (Laverman et al., 2001;
Nugroho et al., 2005, 2007), and vice versa. Nitrification rates were
determined and related to changes in overall bacterial community
profiles, established by 16S rRNA gene-based community profiling.
2. Materials and methods
Table 1
Treatments applied to soils in 6-month incubations. A 10 g fresh weight of native or
sterilised soil samples from Schoorl and Wekerom was placed in 250 ml sterile
bottles, using aseptic techniques, and amended with different inoculants (1 g fresh
soil or 1 ml soil slurry from the same or different site of origin).
Soil condition
Site of origin
Schoorl
Wekerom
Native soil
1. Control
2. Inoculated with 1 g fresh
Wekerom soil
3. Inoculated with 1 ml fresh
Wekerom soil slurry
1. Control
2. Inoculated with 1 g
fresh Schoorl soil
3. Inoculated with 1 ml
fresh Schoorl soil slurry
Sterilised soil
1. Control
2. Inoculated with 1 g fresh
Wekerom soil
3. Inoculated with 1 ml fresh
Wekerom soil slurry
4. Inoculated with 1 g fresh
Schoorl soil
5. Inoculated with 1 ml fresh
Schoorl soil slurry
1. Control
2. Inoculated with 1 g
fresh Schoorl soil
3. Inoculated with 1 ml
fresh Schoorl soil slurry
4. Inoculated with 1 g
fresh Wekerom soil
5. Inoculated with 1 ml
fresh Wekerom soil slurry
2.1. Study sites and soil sampling
The forest floors of Scots pine stands were sampled from Schoorl
(latitude 52 430 N; longitude 4 400 E) and Wekerom (latitude
52 060 N; longitude 5 410 E), The Netherlands. Previously, Nugroho
et al. (2005) determined that net nitrification rates in these soils
1
dry soil wk1, respectively, while
were 0.1 and 14.4 mg NO
3 -N g
1
dry
net ammonification rates were similar (about 23 mg NHþ
4 -N g
1
soil wk ). Further details on the two forest sites used in this study
are given in Nugroho et al. (2005). At each sampling site, eighteen
samples (15 20 cm) of the forest floor (F layer) were randomly
collected from a 5 5 m plot, then randomly pooled to give six
composite samples and returned to the laboratory in cooling boxes.
2.2. Laboratory incubation
Field-moist soils were immediately passed through a 4 mm
sieve in the laboratory and homogenized by hand. Three composite
samples were then stored at 5 C, while three other composite
samples were sterilised by 25 kGy gamma (g-) irradiation at Isotron
Netherland B.V.
Sub-samples of native and sterilised soils were brought to 68%
moisture content (wet weight) by adding sterile demineralised
water. Native or sterilised soil samples (10 g fresh weight) from
Schoorl and Wekerom were put in 250 ml sterile bottles, using
aseptic techniques, and amended with different inoculants (1 g
fresh soil or 1 ml soil slurry from the same or different site of origin)
as outlined in Table 1, each inoculation having 12 replicates. This
inoculation procedure with fresh soil or soil slurry has also been
applied in other studies on microbial-driven processes, including
nitrification (Marschner and Rumberger, 2004; Meier et al., 2006).
Soil slurry was chosen as a treatment in order to add a fraction of
soil microorganisms separate from large soil materials, possibly
containing abiotic factors affecting nitrification. Soil slurries were
prepared by mixing unsterilised soil and sterile 0.1% sodium
pyrophosphate (soil:solution ratio 1:1), shaken for 2 h on a shaker
(100 rev min1) at room temperature: the mixture was allowed to
settle for 15 min before the supernatant containing the desorbed
bacterial cells and small soil particles was decanted into sterile
Eppendorf tubes. Soils were thoroughly homogenized with a sterile
spatula after addition of soil or soil slurry. Bottles were sealed with
cotton plugs and incubated at 18 C in the dark. Soil moisture was
maintained by periodic addition of sterile demineralised water.
Destructive samplings were conducted after 0, 1, 3 and 6 months.
Three bottles were sampled per treatment and per sampling
occasion. Extraction and determination of NHþ
4 -N and NO3 -N
concentrations were carried out as described previously (Nugroho
et al., 2005).
2.3. DNA extraction, PCR, DGGE and cloning
Samples for analysis of bacterial communities were taken at the
end (6 months) of the experiment for each of the triplicate of each
treatment. DNA was extracted from approximately 0.15 g (fresh
weight) sub-samples of soil using the FastDNAÒ SPIN Kit for Soil
(Qbiogene, Carlsbad, CA, USA). The extracted DNA was cleaned with
the Wizard DNA clean-up system (Promega, Madison, WI, USA).
Bacterial 16S rRNA gene fragments were amplified from DNA
extracts in 50 ml reactions containing 400 nM general eubacterial
357F-GC/518R primers (Muyzer et al., 1993), 0.2 mM dNTPs, 10 mg
BSA, 2.5 units Taq DNA polymerase, the buffer conditions recommended by the manufacturer, and 5 ml template. The reaction
conditions were 4 min at 94 C followed by 35 cycles of 30 s at
94 C, 1 min at 54 C, 1 min at 72 C, and 5 min at 72 C for the last
cycle. DGGE (BIO-RAD DcodeÔ systems, Hercules, California, USA)
of 16S rRNA gene fragments was performed using polyacrylamide
gel with a gradient of 30–55% denaturant and run for 4 h at 200 V in
1 TAE buffer at a constant temperature of 60 C. DNA was
visualised after SYBR Gold (Molecular Probes) staining by UV
transilluminating and photographed with a digital camera. To aid
statistical analysis of gels, a marker was added on the outsides and
in the middle of the gels (Ro¨ling et al., 2001).
2.4. Data analysis
A general linear model with type IV sums of squares (Shaw and
Mitchell-Olds, 1993) was used to test the effects of irradiation, site
of origin and inoculation on the initial NHþ
4 -N and NO3 -N
concentrations and pH values using the software SPSS 11.5. This
type of analysis allows for unbalanced design, which is necessary
since there were ten treatment combinations (five levels of treatment factor inoculation and two levels of treatment factor site of
origin) applied to sterilised soil while there were six treatment
combinations (three levels of treatment factor inoculation and two
levels of treatment factor site of origin) applied to native soil. When
required, variables were log10 transformed to fulfill ANOVA
assumptions. Cumulative net mineralisation and nitrification were
calculated by subtracting the initial (NHþ
4 þ NO3 )-N and NO3 -N
concentrations at the start of the experiment from the
(NHþ
4 þ NO3 )-N and NO3 -N concentrations in the soil during the
respective incubation period (1, 3 and 6 months).
245
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
DGGE gel images were converted, normalized and analysed with
the GelCompar 4.0 software package (Applied Maths, Kortrijk,
Belgium) per gel. Similarities between lanes were calculated by
using the Pearson product moment correlation coefficient derived
from the same software package. As each of the triplicate per
treatment was run in a separate DGGE, three similarity values were
obtained for each comparison of two different treatments. These
values were used to test the hypotheses that (i) the origin of
inoculum determines the bacterial community in sterilised soil, or
(ii) the origin of sterilised soil determines the bacterial community
in sterilised soil. These hypotheses were tested by classifying the
similarity coefficients into discrete groups, which were subsequently tested statistically to determine whether their averages
were significantly different (van Verseveld and Ro¨ling, 2004). As
the similarity data were not normally distributed, a non-parametric
analysis (Mann–Whitney U test) was performed using Systat 7.0
(SPSS Inc., Chicago, Illinois). Within-group similarity coefficients
were assigned to testing variable 1 and between-group similarity
coefficients testing variable 2. As an example, for hypothesis (i), all
comparisons among sterilised treatments inoculated with the same
soil were assigned to testing variable 1 while all similarity coefficients comparing a sterilised soil inoculated with Wekerom soil to
a sterilised soil inoculated with Schoorl soil were assigned to
testing variable 2.
A composite matrix was created by averaging the DGGE-based
similarity values of each pair of treatments being compared. The
composite matrix was clustered with the unweighted pair-group
averages (UPGMA) algorithm using PAST program (Hammer et al.,
2001). The variability of DGGE profiles between replicates and the
variability of relationships between treatments were calculated
using coefficient of variation (CV) as standard deviation divided by
the mean of the DGGE-based similarity values of all replicates and
each pair of treatments.
3. Results
3.1. Initial NHþ
4 -N and NO3 -N concentrations and soil pH
In our experiments, 25 kGy g-irradiation significantly (P < 0.05)
increased initial NHþ
4 -N concentration in Schoorl soil from
58.9 mg g1 to 124.4 mg g1, but did not affect the initial NHþ
4 -N
concentration in Wekerom soil significantly (from 120.2 mg g1 to
129.8 mg g1). The initial NO
3 -N concentration in Schoorl soil was
not affected by g-irradiation, but declined (P < 0.05) from
43.6 mg g1 to 8.1 mg g1 in Wekerom soil (Table 2). Initial soil pH
levels in the Schoorl and Wekerom soils were not affected by girradiation (Table 2).
Inoculations of the Schoorl and Wekerom soils with soil or soil
slurry from the same site or the other site did not have a significant
effect on the initial NHþ
4 -N concentrations and initial pH values
(Table 2). On the other hand, initial NO
3 -N concentrations for the
Schoorl soils’ inoculation with Wekerom soil or soil slurry significantly (P < 0.05) increased from 0.1 to 4.3 and 2.8 mg g1, respectively (Table 2). Calculations (data not shown) revealed that these
higher nitrate concentrations can be fully attributed to the fact that
the initial NO
3 -N concentrations in native Wekerom soils are much
higher than in Schoorl soils, and are introduced by the amendment
of the Schoorl soil with native Wekerom soil. Because of the effects
of g-irradiation and cross-inoculation on initial NHþ
4 -N and NO3 -N
concentrations, these values were subtracted from all subsequent
measurements, in order to be able to compare the results of the
different treatments.
3.2. Effects of g-irradiation and inoculants on cumulative net
mineralisation and nitrification in Schoorl soil
Cumulative net mineralisation over the 6-month period in
1
dry
native Schoorl control soil was 149.0 mg (NHþ
4 þ NO3 )-N g
soil (Fig. 1a). When this soil was inoculated with Wekerom soil or
soil slurry, the cumulative net mineralisation was indistinguish
able from the native control soil (182.3 and 149.8 mg (NHþ
4 þ NO3 )1
N g dry soil, respectively (Fig. 1a)). Mineralisation of ammonia
occurred during the first three months in g-irradiated Schoorl
control soil, the cumulative net mineralisation was lower (P < 0.05)
than in native Schoorl control soil, on average 93.8 mg
1
dry soil (Fig. 1b). When this soil was inoculated
(NHþ
4 þ NO3 )-N g
with soil or soil slurry from the same site of origin, this cumulative
net mineralisation increased significantly (P < 0.05) to 212.3 and
1
dry soil, respectively (Fig. 1b). The
203.4 mg (NHþ
4 þ NO3 )-N g
cumulative net mineralisation in g-irradiated Schoorl soil inoculated with Wekerom soil or soil slurry was also significantly
1
dry soil,
(P < 0.05) higher (221.1 and 206.1 mg (NHþ
4 þ NO3 )-N g
respectively) than in the g-irradiated Schoorl control soil (Fig. 1b)
and comparable to the cumulative net mineralisation in native
Schoorl control soil.
Cumulative net nitrification in native Schoorl control soil was
1
dry soil produced over
relatively low, less than 0.5 mg NO
3 -N g
the 6-month period (Fig. 1c). Inoculating the native soil with
Wekerom soil or soil slurry significantly (P < 0.05) and immediately
increased nitrate concentrations, leading to the production of 33.9
1
dry soil in six months, respectively (Fig. 1c).
and 16.3 mg NO
3 -N g
The cumulative net nitrification in g-irradiated Schoorl control soil
was comparable to the native Schoorl control soil (Fig. 1d). Inoculating this g-irradiated soil with Schoorl soil or soil slurry did not
have significant effects on net nitrification, while inoculating this
soil with Wekerom soil or soil slurry again resulted in significantly
(P < 0.05) higher concentrations of nitrate, 27.2 and 10.1 mg NO
3N g1 dry soil, respectively, after six months (Fig. 1d).
Table 2
Initial NHþ
4 -N and NO3 -N concentrations and pH values in Schoorl and Wekerom soils. Standard errors of the mean are shown in brackets, n ¼ 3.
Soil condition
Inoculum
Schoorl
1
NHþ
4 -N (mg g )
Native soil
Control
Soil, allo-inoculateda
Soil slurry, allo-inoculateda
Sterilised soil
Control
Soil, allo-inoculateda
Soil slurry, allo-inoculateda
Soil, auto-inoculatedb
Soil slurry, auto-inoculatedb
58.9 (0.2)
71.2 (0.9)
61.3 (0.3)
124.4
138.3
23.0
127.6
127.9
(2.4)
(4.5)
(2.8)
(5.9)
(5.6)
Wekerom
1
NO
3 -N (mg g )
pHKCl
1
NHþ
4 -N (mg g )
1
NO
3 -N (mg g )
pHKCl
0.3 (0.0)
4.9 (0.7)
3.3 (0.2)
2.8 (0.0)
2.8 (0.0)
2.8 (0.0)
120.2 (12.0)
122.3 (9.7)
115.2 (9.5)
43.6 (6.0)
44.1 (4.6)
44.6 (5.9)
2.9 (0.0)
2.8 (0.0)
2.9 (0.0)
0.1
4.3
2.8
0.2
0.5
2.8
2.8
2.9
2.8
2.8
129.8
136.9
134.0
149.2
138.9
8.1
8.2
8.5
11.4
9.5
2.7
2.7
2.7
2.7
2.7
(0.0)
(0.6)
(0.2)
(0.1)
(0.0)
(0.0)
(0.1)
(0.1)
(0.1)
(0.1)
(1.2)
(1.4)
(3.0)
(3.7)
(0.5)
(0.5)
(0.5)
(0.6)
(0.7)
(0.2)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
a
Soils were inoculated with soil or soil slurry from the other location sampled in this study, sterilised or native Schoorl soil was inoculated with Wekerom soil or soil slurry,
while sterilised or native Wekerom soil was inoculated with Schoorl soil or soil slurry.
b
Soils were inoculated with soil or soil slurry from the same site of origin, sterilised Schoorl soil was inoculated with Schoorl soil or soil slurry, while sterilised Wekerom soil
was inoculated with Wekerom soil or soil slurry.
246
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
a
Native Schoorl soil
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
50
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
300
250
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
c
Native Schoorl soil
200
150
100
50
0
40
30
20
10
0
-10
0
1
2
3
4
5
6
7
0
1
Incubation time (months)
b
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
250
3
4
5
6
7
6
7
Sterilised Schoorl soil
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
50
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
d
Sterilised Schoorl soil
300
2
Incubation time (months)
200
150
100
50
0
40
30
20
10
0
-10
0
1
2
3
4
5
6
Incubation time (months)
7
0
1
2
3
4
5
Incubation time (months)
Fig. 1. Cumulative net mineralisation (a and b) and nitrification (c and d) in native (a and c) and sterilised Schoorl soils (b and d). Bars show standard errors of the mean, n ¼ 3.
3.3. Effects of g-irradiation and inoculants on cumulative net
mineralisation and nitrification in Wekerom soil
Cumulative net mineralisation in native Wekerom control soil
1
dry soil in six months (Fig. 2a).
was 133.9 mg (NHþ
4 þ NO3 )-N g
Inoculation with Schoorl soil or soil slurry significantly (P < 0.05)
increased the cumulative net mineralisation to 158.8 and 157.1 mg
1
dry soil, respectively (Fig. 2a). The cumulative
(NHþ
4 þ NO3 )-N g
net mineralisation in g-irradiated Wekerom control soil was
1
dry soil (Fig. 2b), comparable to native
138.7 mg (NHþ
4 þ NO3 )-N g
Wekerom control soil. When this soil was inoculated with
Wekerom soil or soil slurry, the cumulative net mineralisation
increased significantly (P < 0.05) to 249.3 and 241.3 mg
1
dry soil, respectively (Fig. 2b). The cumulative
(NHþ
4 þ NO3 )-N g
net mineralisation in g-irradiated Wekerom soil inoculated with
Schoorl soil or slurry also increased significantly (P < 0.05) to 240.3
1
dry soil, respectively (Fig. 2b).
and 231.6 mg (NHþ
4 þ NO3 )-N g
Cumulative net nitrification in native Wekerom control soil was
1
dry soil (Fig. 2c). Inoculating this soil with
27.9 mg NO
3 -N g
Schoorl soil or soil slurry significantly (P < 0.05) increased cumu1
dry soil,
lative net nitrification to 37.4 and 41.7 mg NO
3 -N g
respectively (Fig. 2c). The cumulative net nitrification in g-irradiated Wekerom control soil was relatively low, less than 0.5 mg NO
3N g1 dry soil (Fig. 2d). Inoculating this soil with soil from Wekerom
significantly (P < 0.05) increased cumulative net nitrification to
1
dry soil, while inoculating this soil with soil slurry
6.4 mg NO
3 -N g
from Wekerom, or soil and soil slurry from Schoorl did not have
significant effects on the cumulative net nitrification (Fig. 2d).
The effects of g-irradiation and inoculation of Schoorl and
Wekerom soils on soil pH values were not significant. Soil pH values
increased slightly from 2.8 to 3.0 within three months in all
microcosms, irrespective of whether they were sterilised or not,
and stayed the same till the end of the experiment (data not
shown).
3.4. Effects of g-irradiation and inoculants on general bacterial
community profiles in Schoorl and Wekerom soils
Bacteria-specific PCR-DGGE fingerprinting for each of the triplicates per treatment revealed very reproducible profiles, with the
exception of the uninoculated sterilised soils which showed more
variation (Fig. 3). The variability of DGGE profiles between
247
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
a
300
Native Wekerom soil
50
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
c
Native Wekerom soil
250
200
150
100
50
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
40
30
20
10
0
-10
0
0
1
2
3
4
5
6
0
7
1
b
3
4
5
6
7
Sterilised Wekerom soil
50
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
d
Sterilised Wekerom soil
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
300
2
Incubation time (months)
Incubation time (months)
250
200
150
100
50
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
40
30
20
10
0
-10
0
0
1
2
3
4
5
6
Incubation time (months)
7
0
1
2
3
4
5
6
7
Incubation time (months)
Fig. 2. Cumulative net mineralisation (a and b) and nitrification (c and d) in native (a and c) and sterilised Wekerom soils (b and d). Bars show standard errors of the mean, n ¼ 3.
replicates and relationships between treatments was low; the
coefficient of variance (CV) in the percentage of similarity in
community profiles was around 10%. Cluster analysis of
a composite matrix, containing an averaged similarity value per
comparison of two treatments, revealed that bacterial community
structure at the end of the experiment (6 months) revealed
a grouping based on the combination of origin of the soil and type
of treatment (Fig. 4). Sterilisation plus subsequent inoculation had
pronounced impacts on the bacterial community structure in both
soils, in comparison to the native soils (Figs. 3 and 4). The
community fingerprints of sterilised Schoorl and Wekerom control
soils, that did not receive an inoculum, differed most strongly
(similarity of 37.0 mg g1 dry soil month1)
than in the native Wekerom control (27.9 mg g1 dry soil month1),
while the bacterial community structure remained highly similar to
that in the native Wekerom control. Overall, this study indicates that
biotic factors contribute to why some acidic Scots pine forest soils
nitrify and some do not.
Recent research has revealed that ammonia-oxidising Archaea
(AOA) are widespread, abundant and active in grassland soil
(Treusch et al., 2005) and in pristine and agricultural soils, with pH
ranging from 5.5 to 7.3 (Leininger et al., 2006) thus in soils with
higher pHs than the ones studied here. The assumption that AOA
are important under extreme conditions such as acidic conditions
(Valentine, 2007) is not supported by the absence of nitrification in
the acidic Schoorl soil, which suggests the absence or low abundance of AOA, like we also observed for AOB (Nugroho et al., 2005,
2007). However, even if AOA were to be detected in acid soils, their
presence cannot offer an explanation on why some forest soils
nitrify, while others do not. AOB were detected in all forest soils
that revealed high nitrification rates, while in forest soils with low
nitrification AOB were not detectable (Nugroho et al., 2005). It
might therefore be that the same factors that affect AOB and their
nitrifying activity also affect AOA.
Acknowledgements
The authors gratefully acknowledge Frans Kuenen and Rudo
Verweij for field assistance.
References
Balser, T.C., Firestone, M.K., 2005. Linking microbial community composition and
soil processes in a California annual grassland and mixed-conifer forest.
Biogeochemistry 73, 395–415.
Ba¨ckman, J.S.K., Hermansson, A., Tebbe, C.C., Lindgren, P.-E., 2003. Liming induced
growth of a diverse flora of ammonia-oxidising bacteria in acid spruce forest
soil as determined by SSCP and DGGE. Soil Biology & Biochemistry 35,
1337–1347.
Bell, T., Newman, J.A., Silverman, B.W., Turner, S.L., Lilley, A.K., 2005. The contribution of species richness and composition to bacterial services. Nature 436,
1157–1160.
Bottomley, P.J., Taylor, A.E., Boyle, S.A., McMahon, S.K., Rich, J.J., Cromack Jr., K.,
Myrold, D.D., 2004. Responses of nitrification and ammonia-oxidizing bacteria
to reciprocal transfers of soil between adjacent coniferous forest and meadow
vegetation in the Cascade Mountains of Oregon. Microbial Ecology 48, 500–508.
Booth, M.S., Stark, J.M., Rastetter, E., 2005. Controls on nitrogen cycling in terrestrial
ecosystems: a synthetic analysis of literature data. Ecological Monographs 75,
139–157.
250
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
Compton, J.E., Watrud, L.S., Porteous, L.A., DeGrood, S., 2004. Response of soil
microbial biomass and community composition to chronic nitrogen additions at
Harvard forest. Forest Ecology and Management 196, 143–158.
de Boer, W., Gunnewiek, P.J.A.K., Parkinson, D., 1996. Variability of N mineralization
and nitrification in a simple, simulated microbial forest soil community. Soil
Biology & Biochemistry 28, 203–211.
de Boer, W., Kester, R.A., 1996. Variability of nitrification potentials in patches of
undergrowth vegetation in primary Scots pine stands. Forest Ecology and
Management 86, 97–103.
Griffiths, B.S., Kuan, H.L., Ritz, K., Glover, L.A., McCaig, A.E., Fenwick, C., 2004. The
relationship between microbial community structure and functional stability,
tested experimentally in an upland pasture soil. Microbial Ecology 47,
104–113.
Griffiths, B.S., Ritz, K., Bardgett, R.D., Cook, R., Ekelund, F., Sørensen, S.J., Bååth, E.,
Bloem, J., de Ruiter, P.C., Dolfing, J., Nicolardot, B., 2000. Ecosystem response of
pasture soil communities to fumigation-induced microbial diversity reductions:
an examination of the biodiversity–ecosystem function relationship. Oikos 90,
279–294.
Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software
package for education and data analysis. Palaeontologia Electronica 4 (1), 9.
Jones, H.E., West, H.M., Chamberlain, P.M., Parekh, N.R., Beresford, N.A., Crout, N.M.J.,
2004. Effects of gamma irradiation on Holcus lanatus (Yorkshire fog grass) and
associated soil microorganisms. Journal of Environmental Radioactivity 74,
57–71.
Laverman, A.M., Braster, M., Ro¨ling, W.F.M., van Verseveld, H.W., 2005. Bacterial
community structure and metabolic profiles in a forest soil exhibiting
spatially variable net nitrate production. Soil Biology & Biochemistry 37,
1581–1588.
Laverman, A.M., Speksnijder, A.G.C.L., Braster, M., Kowalchuk, G.A., Verhoef, H.A.,
van Verseveld, H.W., 2001. Spatiotemporal stability on an ammonia-oxidizing
community in a nitrogen-saturated forest soil. Microbial Ecology 42, 35–45.
Laverman, A.M., Zoomer, H.R., van Verseveld, H.W., Verhoef, H.A., 2000. Temporal
and spatial variation of nitrogen transformations in a coniferous forest soil. Soil
Biology & Biochemistry 32, 1661–1670.
Leininger, S., Urich, T., Schloter, M., Schwark, L., Qi, J., Nicol, G.W., Prosser, J.I.,
Schuster, S.C., Schleper, C., 2006. Archaea predominate among ammoniaoxidizing prokaryotes in soils. Nature 442, 806–809.
Marschner, P., Rumberger, A., 2004. Rapid changes in the rhizosphere bacterial
community structure during re-colonization of sterilized soil. Biology and
Fertility of Soils 40, 1–6.
McLaren, A.D., 1969. Radiation as a technique in soil biology and biochemistry. Soil
Biology & Biochemistry 1, 63–73.
McNamara, N.P., Black, H.I.J., Beresford, N.A., Parekh, N.R., 2003. Effects of acute
gamma irradiation on chemical, physical and biological properties of soils.
Applied Soil Ecology 24, 117–132.
Meier, E.A., Thorburn, P.J., Probert, M.E., 2006. Occurrence and simulation of nitrification in two contrasting sugarcane soils from the Australian wet tropics.
Australian Journal of Soil Research 44, 1–9.
Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase
chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695–700.
Nugroho, R.A., Ro¨ling, W.F.M., Laverman, A.M., Verhoef, H.A., 2005. Presence of
Nitrosospira cluster 2 bacteria corresponds to N transformation rates in nine
acid Scots pine forest soils. FEMS Microbiology Ecology 53, 472–481.
Nugroho, R.A., Ro¨ling, W.F.M., Laverman, A.M., Verhoef, H.A., 2007. Low nitrification
rates in acid Scots pine forest soils are due to pH-related factors. Microbial
Ecology 53, 89–97.
Persson, T., Wire´n, A., 1995. Nitrogen mineralization and potential nitrification at
different depths in acid forest soil. Plant and Soil 168/169, 55–65.
Ro¨ling, W.F.M., van Breukelen, B.M., Braster, M., Lin, B., van Verseveld, H.W., 2001.
Relationships between microbial community structure and hydrochemistry in
a landfill leachate-polluted aquifer. Applied and Environmental Microbiology
67, 4619–4629.
Shaw, R.G., Mitchell-Olds, T., 1993. ANOVA for unbalanced data: an overview.
Ecology 74, 1638–1645.
Thompson, J.P., 1990. Soil sterilization methods to show VA-mycorrhizae aid P and
Zn nutrition of wheat in vertisols. Soil Biology & Biochemistry 22, 229–240.
Tolli, J., King, G.M., 2005. Diversity and structure of bacterial chemolithotrophic
communities in pine forest and agroecosystem soils. Applied and Environmental Microbiology 71, 8411–8418.
Treusch, A.H., Leininger, S., Kletzin, A., Schuster, S.C., Klenk, H.-P., Schleper, C., 2005.
Novel genes for nitrite reductase and Amo-related proteins indicate a role of
uncultivated mesophilic crenarchaeota in nitrogen cycling. Environmental
Microbiology 7, 1985–1995.
Valentine, D.L., 2007. Adaptations to energy stress dictate the ecology and evolution
of the Archaea. Nature Reviews Microbiology 5, 316–323.
van Niel, E.W.J., Arts, P.A.M., Wesselink, B.J., Robertson, L.A., Kuenen, J.G., 1993.
Competition between heterotrophic and autotrophic nitrifiers for ammonia in
chemostat cultures. FEMS Microbiology Ecology 102, 109–118.
van Verseveld, H.W., Ro¨ling, W.F.M., 2004. Cluster analysis and statistical comparison of molecular community profile data. In: Kowalchuk, G.A., de Bruijn, F.J.,
Head, J.M., Akkermans, A.D.L., van Elsas, J.D. (Eds.)Molecular Microbial Ecology
Manual, second ed., vol. 1.7.4, pp. 1–24.
Verhagen, F.J.M., Duyts, H., Laanbroek, H.J., 1992. Competition for ammonium
between nitrifying and heterotrophic bacteria in continuously percolated soil
columns. Applied and Environmental Microbiology 58, 3303–3311.
Verhagen, F.J.M., Laanbroek, H.J., 1991. Competition for ammonium between nitrifying and heterotrophic bacteria in dual energy-limited chemostats. Applied
and Environmental Microbiology 57, 3255–3263.
Verhagen, F.J.M., Laanbroek, H.J., Woldendorp, J.W., 1995. Competition for ammonium between plant roots and nitrifying and heterotrophic bacteria and the
effects of protozoan grazing. Plant and Soil 170, 241–250.
Wheatley, R.E., Caul, S., Crabb, D., Daniell, T.J., Griffiths, B.S., Ritz, K., 2003. Microbial
population dynamics related to temporal variations in nitrification in three
arable fields. European Journal of Soil Science 54, 707–714.
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Changes in nitrification and bacterial community structure upon crossinoculation of Scots pine forest soils with different initial nitrification rates
Rully A. Nugroho a, Wilfred F.M. Ro¨ling b, *, Nico M. van Straalen a, Herman A. Verhoef a
a
b
Institute of Ecological Science, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
Department of Molecular Cell Physiology, Faculty of Earth and Life Sciences, VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 May 2008
Received in revised form
13 October 2008
Accepted 17 October 2008
Available online 12 November 2008
Nitrification occurs slowly in many acid Scots pine forest soils. We examined if bacterial community
structure and interactions between members of the bacterial community in these forest soils prohibit
growth of ammonia-oxidising microorganisms and their nitrifying activity. Native and gamma-irradiated
Scots pine forest soils known to have low net nitrification rates were augmented with fresh soils or soil
slurries from nitrifying Scots pine forest soil, and vice versa. Augmentation of native non-nitrifying soils
with nitrifying soils induced net nitrification, although no significant changes in bacterial community
structure, as measured by 16S rRNA gene-based denaturing gradient gel electrophoresis (DGGE), were
observed. In sterilised soils, the inoculum, i.e. native nitrifying soil or non-nitrifying soil, determined the
occurrence of net nitrification and bacterial community structure, and not the origin of the sterilised
soils. Our results demonstrate that low net nitrification rates in acid Scots pine forest soils cannot be
(solely) explained by unfavourable abiotic soil conditions, but that still uncaptured biotic factors
contribute to suppression of nitrification.
Ó 2008 Elsevier Ltd. All rights reserved.
keywords:
Cross-inoculation
Bioaugmentation
Net nitrification
Bacterial communities
Scots pine
1. Introduction
Nitrification tests using soil incubations have revealed that
nitrification occurred readily in some acid pine forest soils, but
slowly in others (Bottomley et al., 2004; Nugroho et al., 2005,
2007). Differences in the occurrence and nitrification rate can in
part be explained by abiotic factors. Acid forest soils with low net
nitrification rates are correlated with high C/N ratios (or low total
N) and low atmospheric N depositions (Ba¨ckman et al., 2003;
Compton et al., 2004; Nugroho et al., 2005, 2007; Persson and
Wire´n, 1995; Tolli and King, 2005). Low net nitrification rates in
acid forest soils cannot be explained solely by soil pH, since soil pH
values did not differ significantly between soils with low and high
net nitrification rates (Nugroho et al., 2005). N availability also does
not constrain net nitrification in these soils as large amounts of
NHþ
4 -N are produced during incubation of these soils in the laboratory (Booth et al., 2005; Nugroho et al., 2005; Persson and Wire´n,
1995). As in some acid soils with increased NHþ
4 -N concentrations
net nitrification does not occur (Nugroho et al., 2007), other
suppressive factors need to be considered.
Differences in nitrification could also be driven by biotic factors.
Nitrification rates might be linked to the composition of the soil
* Corresponding author. Tel.: þ31 20 598 7192; fax: þ31 20 598 7229.
E-mail address: wilfred.roling@falw.vu.nl (W.F.M. Ro¨ling).
0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2008.10.020
bacterial community (Balser and Firestone, 2005; de Boer and Kester, 1996; de Boer et al., 1996; Wheatley et al., 2003). Community
members can have specific positive or negative effects on the growth
and activity of the nitrifying bacteria. Chitinolytic soil bacteria can
produce antibiotics against nitrifying bacteria (de Boer et al., 1996).
Ammonia-oxidising bacteria (AOB) are poor competitors for NHþ
4
relative to ammonia-assimilating heterotrophs when ammonium is
limiting (van Niel et al., 1993; Verhagen and Laanbroek, 1991; Verhagen et al., 1992, 1995). Wheatley et al. (2003), using a nested PCR
with the not very specific CTO primer set, also indicated that nitrification rates might be linked to the composition of the soil bacterial
community, rather than to the AOB community itself. They
compared the structure of the bacterial community within and
between three arable fields differing in potential nitrification rates
but broadly similar in basic characteristics (soil pH, total C and total
N contents). The bacterial community structure in each field differed
significantly. In contrast, molecular analyses specific to AOB suggested that the populations in all three fields were similar in types
and did not vary with time (Wheatley et al., 2003).
The relationships between overall bacterial community structure and nitrification rates in acid Scots pine forest soils have not
been studied thoroughly. Previous studies (Laverman et al., 2000;
Nugroho et al., 2005), applying specific inhibitors of autotrophic
nitrification, have revealed that heterotrophic nitrification does not
play a significant role in such soils. Our aim was to examine if
general bacterial community structure and interactions between
244
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
members of the bacterial community in acid Scots pine forest soils
with low nitrification rate prohibit growth of ammonia oxidizers
and their nitrifying activity. If so, we may expect that when native
soils (showing either low or high nitrification rates) are inoculated,
or augmented, with a small quantity of forest soil with contrasting
nitrification activity, no significant changes in nitrification will
occur. However, we would expect that when the soil microbial
community is destroyed by sterilisation, the nitrifying potential of
the inoculum will be able to establish itself in the sterilised soil,
unless abiotic conditions in the sterilised soil prohibit this. To test
these hypotheses, native and gamma-irradiated Schoorl (The
Netherlands) soils, with low nitrification rates (Nugroho et al.,
2005, 2007), were augmented with fresh soils or soil slurries from
nitrifying Wekerom soil (The Netherlands) (Laverman et al., 2001;
Nugroho et al., 2005, 2007), and vice versa. Nitrification rates were
determined and related to changes in overall bacterial community
profiles, established by 16S rRNA gene-based community profiling.
2. Materials and methods
Table 1
Treatments applied to soils in 6-month incubations. A 10 g fresh weight of native or
sterilised soil samples from Schoorl and Wekerom was placed in 250 ml sterile
bottles, using aseptic techniques, and amended with different inoculants (1 g fresh
soil or 1 ml soil slurry from the same or different site of origin).
Soil condition
Site of origin
Schoorl
Wekerom
Native soil
1. Control
2. Inoculated with 1 g fresh
Wekerom soil
3. Inoculated with 1 ml fresh
Wekerom soil slurry
1. Control
2. Inoculated with 1 g
fresh Schoorl soil
3. Inoculated with 1 ml
fresh Schoorl soil slurry
Sterilised soil
1. Control
2. Inoculated with 1 g fresh
Wekerom soil
3. Inoculated with 1 ml fresh
Wekerom soil slurry
4. Inoculated with 1 g fresh
Schoorl soil
5. Inoculated with 1 ml fresh
Schoorl soil slurry
1. Control
2. Inoculated with 1 g
fresh Schoorl soil
3. Inoculated with 1 ml
fresh Schoorl soil slurry
4. Inoculated with 1 g
fresh Wekerom soil
5. Inoculated with 1 ml
fresh Wekerom soil slurry
2.1. Study sites and soil sampling
The forest floors of Scots pine stands were sampled from Schoorl
(latitude 52 430 N; longitude 4 400 E) and Wekerom (latitude
52 060 N; longitude 5 410 E), The Netherlands. Previously, Nugroho
et al. (2005) determined that net nitrification rates in these soils
1
dry soil wk1, respectively, while
were 0.1 and 14.4 mg NO
3 -N g
1
dry
net ammonification rates were similar (about 23 mg NHþ
4 -N g
1
soil wk ). Further details on the two forest sites used in this study
are given in Nugroho et al. (2005). At each sampling site, eighteen
samples (15 20 cm) of the forest floor (F layer) were randomly
collected from a 5 5 m plot, then randomly pooled to give six
composite samples and returned to the laboratory in cooling boxes.
2.2. Laboratory incubation
Field-moist soils were immediately passed through a 4 mm
sieve in the laboratory and homogenized by hand. Three composite
samples were then stored at 5 C, while three other composite
samples were sterilised by 25 kGy gamma (g-) irradiation at Isotron
Netherland B.V.
Sub-samples of native and sterilised soils were brought to 68%
moisture content (wet weight) by adding sterile demineralised
water. Native or sterilised soil samples (10 g fresh weight) from
Schoorl and Wekerom were put in 250 ml sterile bottles, using
aseptic techniques, and amended with different inoculants (1 g
fresh soil or 1 ml soil slurry from the same or different site of origin)
as outlined in Table 1, each inoculation having 12 replicates. This
inoculation procedure with fresh soil or soil slurry has also been
applied in other studies on microbial-driven processes, including
nitrification (Marschner and Rumberger, 2004; Meier et al., 2006).
Soil slurry was chosen as a treatment in order to add a fraction of
soil microorganisms separate from large soil materials, possibly
containing abiotic factors affecting nitrification. Soil slurries were
prepared by mixing unsterilised soil and sterile 0.1% sodium
pyrophosphate (soil:solution ratio 1:1), shaken for 2 h on a shaker
(100 rev min1) at room temperature: the mixture was allowed to
settle for 15 min before the supernatant containing the desorbed
bacterial cells and small soil particles was decanted into sterile
Eppendorf tubes. Soils were thoroughly homogenized with a sterile
spatula after addition of soil or soil slurry. Bottles were sealed with
cotton plugs and incubated at 18 C in the dark. Soil moisture was
maintained by periodic addition of sterile demineralised water.
Destructive samplings were conducted after 0, 1, 3 and 6 months.
Three bottles were sampled per treatment and per sampling
occasion. Extraction and determination of NHþ
4 -N and NO3 -N
concentrations were carried out as described previously (Nugroho
et al., 2005).
2.3. DNA extraction, PCR, DGGE and cloning
Samples for analysis of bacterial communities were taken at the
end (6 months) of the experiment for each of the triplicate of each
treatment. DNA was extracted from approximately 0.15 g (fresh
weight) sub-samples of soil using the FastDNAÒ SPIN Kit for Soil
(Qbiogene, Carlsbad, CA, USA). The extracted DNA was cleaned with
the Wizard DNA clean-up system (Promega, Madison, WI, USA).
Bacterial 16S rRNA gene fragments were amplified from DNA
extracts in 50 ml reactions containing 400 nM general eubacterial
357F-GC/518R primers (Muyzer et al., 1993), 0.2 mM dNTPs, 10 mg
BSA, 2.5 units Taq DNA polymerase, the buffer conditions recommended by the manufacturer, and 5 ml template. The reaction
conditions were 4 min at 94 C followed by 35 cycles of 30 s at
94 C, 1 min at 54 C, 1 min at 72 C, and 5 min at 72 C for the last
cycle. DGGE (BIO-RAD DcodeÔ systems, Hercules, California, USA)
of 16S rRNA gene fragments was performed using polyacrylamide
gel with a gradient of 30–55% denaturant and run for 4 h at 200 V in
1 TAE buffer at a constant temperature of 60 C. DNA was
visualised after SYBR Gold (Molecular Probes) staining by UV
transilluminating and photographed with a digital camera. To aid
statistical analysis of gels, a marker was added on the outsides and
in the middle of the gels (Ro¨ling et al., 2001).
2.4. Data analysis
A general linear model with type IV sums of squares (Shaw and
Mitchell-Olds, 1993) was used to test the effects of irradiation, site
of origin and inoculation on the initial NHþ
4 -N and NO3 -N
concentrations and pH values using the software SPSS 11.5. This
type of analysis allows for unbalanced design, which is necessary
since there were ten treatment combinations (five levels of treatment factor inoculation and two levels of treatment factor site of
origin) applied to sterilised soil while there were six treatment
combinations (three levels of treatment factor inoculation and two
levels of treatment factor site of origin) applied to native soil. When
required, variables were log10 transformed to fulfill ANOVA
assumptions. Cumulative net mineralisation and nitrification were
calculated by subtracting the initial (NHþ
4 þ NO3 )-N and NO3 -N
concentrations at the start of the experiment from the
(NHþ
4 þ NO3 )-N and NO3 -N concentrations in the soil during the
respective incubation period (1, 3 and 6 months).
245
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
DGGE gel images were converted, normalized and analysed with
the GelCompar 4.0 software package (Applied Maths, Kortrijk,
Belgium) per gel. Similarities between lanes were calculated by
using the Pearson product moment correlation coefficient derived
from the same software package. As each of the triplicate per
treatment was run in a separate DGGE, three similarity values were
obtained for each comparison of two different treatments. These
values were used to test the hypotheses that (i) the origin of
inoculum determines the bacterial community in sterilised soil, or
(ii) the origin of sterilised soil determines the bacterial community
in sterilised soil. These hypotheses were tested by classifying the
similarity coefficients into discrete groups, which were subsequently tested statistically to determine whether their averages
were significantly different (van Verseveld and Ro¨ling, 2004). As
the similarity data were not normally distributed, a non-parametric
analysis (Mann–Whitney U test) was performed using Systat 7.0
(SPSS Inc., Chicago, Illinois). Within-group similarity coefficients
were assigned to testing variable 1 and between-group similarity
coefficients testing variable 2. As an example, for hypothesis (i), all
comparisons among sterilised treatments inoculated with the same
soil were assigned to testing variable 1 while all similarity coefficients comparing a sterilised soil inoculated with Wekerom soil to
a sterilised soil inoculated with Schoorl soil were assigned to
testing variable 2.
A composite matrix was created by averaging the DGGE-based
similarity values of each pair of treatments being compared. The
composite matrix was clustered with the unweighted pair-group
averages (UPGMA) algorithm using PAST program (Hammer et al.,
2001). The variability of DGGE profiles between replicates and the
variability of relationships between treatments were calculated
using coefficient of variation (CV) as standard deviation divided by
the mean of the DGGE-based similarity values of all replicates and
each pair of treatments.
3. Results
3.1. Initial NHþ
4 -N and NO3 -N concentrations and soil pH
In our experiments, 25 kGy g-irradiation significantly (P < 0.05)
increased initial NHþ
4 -N concentration in Schoorl soil from
58.9 mg g1 to 124.4 mg g1, but did not affect the initial NHþ
4 -N
concentration in Wekerom soil significantly (from 120.2 mg g1 to
129.8 mg g1). The initial NO
3 -N concentration in Schoorl soil was
not affected by g-irradiation, but declined (P < 0.05) from
43.6 mg g1 to 8.1 mg g1 in Wekerom soil (Table 2). Initial soil pH
levels in the Schoorl and Wekerom soils were not affected by girradiation (Table 2).
Inoculations of the Schoorl and Wekerom soils with soil or soil
slurry from the same site or the other site did not have a significant
effect on the initial NHþ
4 -N concentrations and initial pH values
(Table 2). On the other hand, initial NO
3 -N concentrations for the
Schoorl soils’ inoculation with Wekerom soil or soil slurry significantly (P < 0.05) increased from 0.1 to 4.3 and 2.8 mg g1, respectively (Table 2). Calculations (data not shown) revealed that these
higher nitrate concentrations can be fully attributed to the fact that
the initial NO
3 -N concentrations in native Wekerom soils are much
higher than in Schoorl soils, and are introduced by the amendment
of the Schoorl soil with native Wekerom soil. Because of the effects
of g-irradiation and cross-inoculation on initial NHþ
4 -N and NO3 -N
concentrations, these values were subtracted from all subsequent
measurements, in order to be able to compare the results of the
different treatments.
3.2. Effects of g-irradiation and inoculants on cumulative net
mineralisation and nitrification in Schoorl soil
Cumulative net mineralisation over the 6-month period in
1
dry
native Schoorl control soil was 149.0 mg (NHþ
4 þ NO3 )-N g
soil (Fig. 1a). When this soil was inoculated with Wekerom soil or
soil slurry, the cumulative net mineralisation was indistinguish
able from the native control soil (182.3 and 149.8 mg (NHþ
4 þ NO3 )1
N g dry soil, respectively (Fig. 1a)). Mineralisation of ammonia
occurred during the first three months in g-irradiated Schoorl
control soil, the cumulative net mineralisation was lower (P < 0.05)
than in native Schoorl control soil, on average 93.8 mg
1
dry soil (Fig. 1b). When this soil was inoculated
(NHþ
4 þ NO3 )-N g
with soil or soil slurry from the same site of origin, this cumulative
net mineralisation increased significantly (P < 0.05) to 212.3 and
1
dry soil, respectively (Fig. 1b). The
203.4 mg (NHþ
4 þ NO3 )-N g
cumulative net mineralisation in g-irradiated Schoorl soil inoculated with Wekerom soil or soil slurry was also significantly
1
dry soil,
(P < 0.05) higher (221.1 and 206.1 mg (NHþ
4 þ NO3 )-N g
respectively) than in the g-irradiated Schoorl control soil (Fig. 1b)
and comparable to the cumulative net mineralisation in native
Schoorl control soil.
Cumulative net nitrification in native Schoorl control soil was
1
dry soil produced over
relatively low, less than 0.5 mg NO
3 -N g
the 6-month period (Fig. 1c). Inoculating the native soil with
Wekerom soil or soil slurry significantly (P < 0.05) and immediately
increased nitrate concentrations, leading to the production of 33.9
1
dry soil in six months, respectively (Fig. 1c).
and 16.3 mg NO
3 -N g
The cumulative net nitrification in g-irradiated Schoorl control soil
was comparable to the native Schoorl control soil (Fig. 1d). Inoculating this g-irradiated soil with Schoorl soil or soil slurry did not
have significant effects on net nitrification, while inoculating this
soil with Wekerom soil or soil slurry again resulted in significantly
(P < 0.05) higher concentrations of nitrate, 27.2 and 10.1 mg NO
3N g1 dry soil, respectively, after six months (Fig. 1d).
Table 2
Initial NHþ
4 -N and NO3 -N concentrations and pH values in Schoorl and Wekerom soils. Standard errors of the mean are shown in brackets, n ¼ 3.
Soil condition
Inoculum
Schoorl
1
NHþ
4 -N (mg g )
Native soil
Control
Soil, allo-inoculateda
Soil slurry, allo-inoculateda
Sterilised soil
Control
Soil, allo-inoculateda
Soil slurry, allo-inoculateda
Soil, auto-inoculatedb
Soil slurry, auto-inoculatedb
58.9 (0.2)
71.2 (0.9)
61.3 (0.3)
124.4
138.3
23.0
127.6
127.9
(2.4)
(4.5)
(2.8)
(5.9)
(5.6)
Wekerom
1
NO
3 -N (mg g )
pHKCl
1
NHþ
4 -N (mg g )
1
NO
3 -N (mg g )
pHKCl
0.3 (0.0)
4.9 (0.7)
3.3 (0.2)
2.8 (0.0)
2.8 (0.0)
2.8 (0.0)
120.2 (12.0)
122.3 (9.7)
115.2 (9.5)
43.6 (6.0)
44.1 (4.6)
44.6 (5.9)
2.9 (0.0)
2.8 (0.0)
2.9 (0.0)
0.1
4.3
2.8
0.2
0.5
2.8
2.8
2.9
2.8
2.8
129.8
136.9
134.0
149.2
138.9
8.1
8.2
8.5
11.4
9.5
2.7
2.7
2.7
2.7
2.7
(0.0)
(0.6)
(0.2)
(0.1)
(0.0)
(0.0)
(0.1)
(0.1)
(0.1)
(0.1)
(1.2)
(1.4)
(3.0)
(3.7)
(0.5)
(0.5)
(0.5)
(0.6)
(0.7)
(0.2)
(0.0)
(0.0)
(0.0)
(0.0)
(0.0)
a
Soils were inoculated with soil or soil slurry from the other location sampled in this study, sterilised or native Schoorl soil was inoculated with Wekerom soil or soil slurry,
while sterilised or native Wekerom soil was inoculated with Schoorl soil or soil slurry.
b
Soils were inoculated with soil or soil slurry from the same site of origin, sterilised Schoorl soil was inoculated with Schoorl soil or soil slurry, while sterilised Wekerom soil
was inoculated with Wekerom soil or soil slurry.
246
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
a
Native Schoorl soil
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
50
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
300
250
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
c
Native Schoorl soil
200
150
100
50
0
40
30
20
10
0
-10
0
1
2
3
4
5
6
7
0
1
Incubation time (months)
b
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
250
3
4
5
6
7
6
7
Sterilised Schoorl soil
Control
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
50
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
d
Sterilised Schoorl soil
300
2
Incubation time (months)
200
150
100
50
0
40
30
20
10
0
-10
0
1
2
3
4
5
6
Incubation time (months)
7
0
1
2
3
4
5
Incubation time (months)
Fig. 1. Cumulative net mineralisation (a and b) and nitrification (c and d) in native (a and c) and sterilised Schoorl soils (b and d). Bars show standard errors of the mean, n ¼ 3.
3.3. Effects of g-irradiation and inoculants on cumulative net
mineralisation and nitrification in Wekerom soil
Cumulative net mineralisation in native Wekerom control soil
1
dry soil in six months (Fig. 2a).
was 133.9 mg (NHþ
4 þ NO3 )-N g
Inoculation with Schoorl soil or soil slurry significantly (P < 0.05)
increased the cumulative net mineralisation to 158.8 and 157.1 mg
1
dry soil, respectively (Fig. 2a). The cumulative
(NHþ
4 þ NO3 )-N g
net mineralisation in g-irradiated Wekerom control soil was
1
dry soil (Fig. 2b), comparable to native
138.7 mg (NHþ
4 þ NO3 )-N g
Wekerom control soil. When this soil was inoculated with
Wekerom soil or soil slurry, the cumulative net mineralisation
increased significantly (P < 0.05) to 249.3 and 241.3 mg
1
dry soil, respectively (Fig. 2b). The cumulative
(NHþ
4 þ NO3 )-N g
net mineralisation in g-irradiated Wekerom soil inoculated with
Schoorl soil or slurry also increased significantly (P < 0.05) to 240.3
1
dry soil, respectively (Fig. 2b).
and 231.6 mg (NHþ
4 þ NO3 )-N g
Cumulative net nitrification in native Wekerom control soil was
1
dry soil (Fig. 2c). Inoculating this soil with
27.9 mg NO
3 -N g
Schoorl soil or soil slurry significantly (P < 0.05) increased cumu1
dry soil,
lative net nitrification to 37.4 and 41.7 mg NO
3 -N g
respectively (Fig. 2c). The cumulative net nitrification in g-irradiated Wekerom control soil was relatively low, less than 0.5 mg NO
3N g1 dry soil (Fig. 2d). Inoculating this soil with soil from Wekerom
significantly (P < 0.05) increased cumulative net nitrification to
1
dry soil, while inoculating this soil with soil slurry
6.4 mg NO
3 -N g
from Wekerom, or soil and soil slurry from Schoorl did not have
significant effects on the cumulative net nitrification (Fig. 2d).
The effects of g-irradiation and inoculation of Schoorl and
Wekerom soils on soil pH values were not significant. Soil pH values
increased slightly from 2.8 to 3.0 within three months in all
microcosms, irrespective of whether they were sterilised or not,
and stayed the same till the end of the experiment (data not
shown).
3.4. Effects of g-irradiation and inoculants on general bacterial
community profiles in Schoorl and Wekerom soils
Bacteria-specific PCR-DGGE fingerprinting for each of the triplicates per treatment revealed very reproducible profiles, with the
exception of the uninoculated sterilised soils which showed more
variation (Fig. 3). The variability of DGGE profiles between
247
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
a
300
Native Wekerom soil
50
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
c
Native Wekerom soil
250
200
150
100
50
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
40
30
20
10
0
-10
0
0
1
2
3
4
5
6
0
7
1
b
3
4
5
6
7
Sterilised Wekerom soil
50
Cumulative net nitrification
(µg NO3--N g-1)
Cumulative net mineralisation
(µg (NH4++NO3-)-N g-1)
d
Sterilised Wekerom soil
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
300
2
Incubation time (months)
Incubation time (months)
250
200
150
100
50
Control
Inoculated with Schoorl soil
Inoculated with Schoorl soil slurry
Inoculated with Wekerom soil
Inoculated with Wekerom soil slurry
40
30
20
10
0
-10
0
0
1
2
3
4
5
6
Incubation time (months)
7
0
1
2
3
4
5
6
7
Incubation time (months)
Fig. 2. Cumulative net mineralisation (a and b) and nitrification (c and d) in native (a and c) and sterilised Wekerom soils (b and d). Bars show standard errors of the mean, n ¼ 3.
replicates and relationships between treatments was low; the
coefficient of variance (CV) in the percentage of similarity in
community profiles was around 10%. Cluster analysis of
a composite matrix, containing an averaged similarity value per
comparison of two treatments, revealed that bacterial community
structure at the end of the experiment (6 months) revealed
a grouping based on the combination of origin of the soil and type
of treatment (Fig. 4). Sterilisation plus subsequent inoculation had
pronounced impacts on the bacterial community structure in both
soils, in comparison to the native soils (Figs. 3 and 4). The
community fingerprints of sterilised Schoorl and Wekerom control
soils, that did not receive an inoculum, differed most strongly
(similarity of 37.0 mg g1 dry soil month1)
than in the native Wekerom control (27.9 mg g1 dry soil month1),
while the bacterial community structure remained highly similar to
that in the native Wekerom control. Overall, this study indicates that
biotic factors contribute to why some acidic Scots pine forest soils
nitrify and some do not.
Recent research has revealed that ammonia-oxidising Archaea
(AOA) are widespread, abundant and active in grassland soil
(Treusch et al., 2005) and in pristine and agricultural soils, with pH
ranging from 5.5 to 7.3 (Leininger et al., 2006) thus in soils with
higher pHs than the ones studied here. The assumption that AOA
are important under extreme conditions such as acidic conditions
(Valentine, 2007) is not supported by the absence of nitrification in
the acidic Schoorl soil, which suggests the absence or low abundance of AOA, like we also observed for AOB (Nugroho et al., 2005,
2007). However, even if AOA were to be detected in acid soils, their
presence cannot offer an explanation on why some forest soils
nitrify, while others do not. AOB were detected in all forest soils
that revealed high nitrification rates, while in forest soils with low
nitrification AOB were not detectable (Nugroho et al., 2005). It
might therefore be that the same factors that affect AOB and their
nitrifying activity also affect AOA.
Acknowledgements
The authors gratefully acknowledge Frans Kuenen and Rudo
Verweij for field assistance.
References
Balser, T.C., Firestone, M.K., 2005. Linking microbial community composition and
soil processes in a California annual grassland and mixed-conifer forest.
Biogeochemistry 73, 395–415.
Ba¨ckman, J.S.K., Hermansson, A., Tebbe, C.C., Lindgren, P.-E., 2003. Liming induced
growth of a diverse flora of ammonia-oxidising bacteria in acid spruce forest
soil as determined by SSCP and DGGE. Soil Biology & Biochemistry 35,
1337–1347.
Bell, T., Newman, J.A., Silverman, B.W., Turner, S.L., Lilley, A.K., 2005. The contribution of species richness and composition to bacterial services. Nature 436,
1157–1160.
Bottomley, P.J., Taylor, A.E., Boyle, S.A., McMahon, S.K., Rich, J.J., Cromack Jr., K.,
Myrold, D.D., 2004. Responses of nitrification and ammonia-oxidizing bacteria
to reciprocal transfers of soil between adjacent coniferous forest and meadow
vegetation in the Cascade Mountains of Oregon. Microbial Ecology 48, 500–508.
Booth, M.S., Stark, J.M., Rastetter, E., 2005. Controls on nitrogen cycling in terrestrial
ecosystems: a synthetic analysis of literature data. Ecological Monographs 75,
139–157.
250
R.A. Nugroho et al. / Soil Biology & Biochemistry 41 (2009) 243–250
Compton, J.E., Watrud, L.S., Porteous, L.A., DeGrood, S., 2004. Response of soil
microbial biomass and community composition to chronic nitrogen additions at
Harvard forest. Forest Ecology and Management 196, 143–158.
de Boer, W., Gunnewiek, P.J.A.K., Parkinson, D., 1996. Variability of N mineralization
and nitrification in a simple, simulated microbial forest soil community. Soil
Biology & Biochemistry 28, 203–211.
de Boer, W., Kester, R.A., 1996. Variability of nitrification potentials in patches of
undergrowth vegetation in primary Scots pine stands. Forest Ecology and
Management 86, 97–103.
Griffiths, B.S., Kuan, H.L., Ritz, K., Glover, L.A., McCaig, A.E., Fenwick, C., 2004. The
relationship between microbial community structure and functional stability,
tested experimentally in an upland pasture soil. Microbial Ecology 47,
104–113.
Griffiths, B.S., Ritz, K., Bardgett, R.D., Cook, R., Ekelund, F., Sørensen, S.J., Bååth, E.,
Bloem, J., de Ruiter, P.C., Dolfing, J., Nicolardot, B., 2000. Ecosystem response of
pasture soil communities to fumigation-induced microbial diversity reductions:
an examination of the biodiversity–ecosystem function relationship. Oikos 90,
279–294.
Hammer, Ø., Harper, D.A.T., Ryan, P.D., 2001. PAST: paleontological statistics software
package for education and data analysis. Palaeontologia Electronica 4 (1), 9.
Jones, H.E., West, H.M., Chamberlain, P.M., Parekh, N.R., Beresford, N.A., Crout, N.M.J.,
2004. Effects of gamma irradiation on Holcus lanatus (Yorkshire fog grass) and
associated soil microorganisms. Journal of Environmental Radioactivity 74,
57–71.
Laverman, A.M., Braster, M., Ro¨ling, W.F.M., van Verseveld, H.W., 2005. Bacterial
community structure and metabolic profiles in a forest soil exhibiting
spatially variable net nitrate production. Soil Biology & Biochemistry 37,
1581–1588.
Laverman, A.M., Speksnijder, A.G.C.L., Braster, M., Kowalchuk, G.A., Verhoef, H.A.,
van Verseveld, H.W., 2001. Spatiotemporal stability on an ammonia-oxidizing
community in a nitrogen-saturated forest soil. Microbial Ecology 42, 35–45.
Laverman, A.M., Zoomer, H.R., van Verseveld, H.W., Verhoef, H.A., 2000. Temporal
and spatial variation of nitrogen transformations in a coniferous forest soil. Soil
Biology & Biochemistry 32, 1661–1670.
Leininger, S., Urich, T., Schloter, M., Schwark, L., Qi, J., Nicol, G.W., Prosser, J.I.,
Schuster, S.C., Schleper, C., 2006. Archaea predominate among ammoniaoxidizing prokaryotes in soils. Nature 442, 806–809.
Marschner, P., Rumberger, A., 2004. Rapid changes in the rhizosphere bacterial
community structure during re-colonization of sterilized soil. Biology and
Fertility of Soils 40, 1–6.
McLaren, A.D., 1969. Radiation as a technique in soil biology and biochemistry. Soil
Biology & Biochemistry 1, 63–73.
McNamara, N.P., Black, H.I.J., Beresford, N.A., Parekh, N.R., 2003. Effects of acute
gamma irradiation on chemical, physical and biological properties of soils.
Applied Soil Ecology 24, 117–132.
Meier, E.A., Thorburn, P.J., Probert, M.E., 2006. Occurrence and simulation of nitrification in two contrasting sugarcane soils from the Australian wet tropics.
Australian Journal of Soil Research 44, 1–9.
Muyzer, G., de Waal, E.C., Uitterlinden, A.G., 1993. Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase
chain reaction-amplified genes coding for 16S rRNA. Applied and Environmental Microbiology 59, 695–700.
Nugroho, R.A., Ro¨ling, W.F.M., Laverman, A.M., Verhoef, H.A., 2005. Presence of
Nitrosospira cluster 2 bacteria corresponds to N transformation rates in nine
acid Scots pine forest soils. FEMS Microbiology Ecology 53, 472–481.
Nugroho, R.A., Ro¨ling, W.F.M., Laverman, A.M., Verhoef, H.A., 2007. Low nitrification
rates in acid Scots pine forest soils are due to pH-related factors. Microbial
Ecology 53, 89–97.
Persson, T., Wire´n, A., 1995. Nitrogen mineralization and potential nitrification at
different depths in acid forest soil. Plant and Soil 168/169, 55–65.
Ro¨ling, W.F.M., van Breukelen, B.M., Braster, M., Lin, B., van Verseveld, H.W., 2001.
Relationships between microbial community structure and hydrochemistry in
a landfill leachate-polluted aquifer. Applied and Environmental Microbiology
67, 4619–4629.
Shaw, R.G., Mitchell-Olds, T., 1993. ANOVA for unbalanced data: an overview.
Ecology 74, 1638–1645.
Thompson, J.P., 1990. Soil sterilization methods to show VA-mycorrhizae aid P and
Zn nutrition of wheat in vertisols. Soil Biology & Biochemistry 22, 229–240.
Tolli, J., King, G.M., 2005. Diversity and structure of bacterial chemolithotrophic
communities in pine forest and agroecosystem soils. Applied and Environmental Microbiology 71, 8411–8418.
Treusch, A.H., Leininger, S., Kletzin, A., Schuster, S.C., Klenk, H.-P., Schleper, C., 2005.
Novel genes for nitrite reductase and Amo-related proteins indicate a role of
uncultivated mesophilic crenarchaeota in nitrogen cycling. Environmental
Microbiology 7, 1985–1995.
Valentine, D.L., 2007. Adaptations to energy stress dictate the ecology and evolution
of the Archaea. Nature Reviews Microbiology 5, 316–323.
van Niel, E.W.J., Arts, P.A.M., Wesselink, B.J., Robertson, L.A., Kuenen, J.G., 1993.
Competition between heterotrophic and autotrophic nitrifiers for ammonia in
chemostat cultures. FEMS Microbiology Ecology 102, 109–118.
van Verseveld, H.W., Ro¨ling, W.F.M., 2004. Cluster analysis and statistical comparison of molecular community profile data. In: Kowalchuk, G.A., de Bruijn, F.J.,
Head, J.M., Akkermans, A.D.L., van Elsas, J.D. (Eds.)Molecular Microbial Ecology
Manual, second ed., vol. 1.7.4, pp. 1–24.
Verhagen, F.J.M., Duyts, H., Laanbroek, H.J., 1992. Competition for ammonium
between nitrifying and heterotrophic bacteria in continuously percolated soil
columns. Applied and Environmental Microbiology 58, 3303–3311.
Verhagen, F.J.M., Laanbroek, H.J., 1991. Competition for ammonium between nitrifying and heterotrophic bacteria in dual energy-limited chemostats. Applied
and Environmental Microbiology 57, 3255–3263.
Verhagen, F.J.M., Laanbroek, H.J., Woldendorp, J.W., 1995. Competition for ammonium between plant roots and nitrifying and heterotrophic bacteria and the
effects of protozoan grazing. Plant and Soil 170, 241–250.
Wheatley, R.E., Caul, S., Crabb, D., Daniell, T.J., Griffiths, B.S., Ritz, K., 2003. Microbial
population dynamics related to temporal variations in nitrification in three
arable fields. European Journal of Soil Science 54, 707–714.