Soil Biology & Biochemistry 40 (2008) 2334–2343

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Antagonistic and synergistic effects of fungal and bacterial growth in soil after
adding different carbon and nitrogen sources
Sandra Meidute, Fredrik Demoling, Erland Bååth*
Department of Microbial Ecology, Lund University, Ecology Building, SE-223 62 Lund, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: The effect of adding easily available and more complex carbon sources, with and without nitrogen, on
Received 8 January 2008 fungal and bacterial growth and activity in soil were studied in the laboratory. Total microbial activity
Received in revised form 29 April 2008 was estimated by measuring respiration, fungal growth with the acetate-in-ergosterol incorporation
Accepted 13 May 2008
technique and bacterial growth with the thymidine and leucine incorporation techniques. The substrate
Available online 11 June 2008
additions consisted of glucose and cellulose, with and without nitrogen (as ammonium nitrate), and
gelatine. The microbial development was followed over a 2-month period. The respiration rate increased
within a few days after adding glucose, with and without nitrogen, and gelatine, initially by more than 10
Keywords:
Fungi
times, but after 2 months no differences were seen compared with the control. Bacterial growth esti-
Bacteria mated with the thymidine and leucine incorporation techniques gave similar results. Adding glucose
Thymidine with nitrogen, or gelatine, increased bacterial growth within a few days up to 10 times, but even after 2
Leucine months of incubation bacterial growth rates were still about 5 times higher than in the control. Adding
14
C acetate only glucose increased bacterial growth rates by about twice over the whole incubation period. Fungal
Ergosterol growth rates especially increased after adding cellulose and nitrogen, although a minor increase was
found after adding cellulose alone. Fungal growth rates started to increase after 10 days of incubation
with cellulose. There were indications of synergistic effects in that bacterial growth increased after the
fungi had started to grow after adding cellulose. Treatments resulting in high bacterial growth rates
(adding easily available carbon sources) led to decreased fungal growth rates compared with the control,
indicating antagonistic effects of bacteria.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction bacterial or fungal decomposition can result in different amounts

Bacteria and fungi are the dominating groups of organisms
found in soil with regard to both biomass and metabolic activity.
Although most fungi and bacteria in soil are decomposer organ-
isms, some substrates are reported to be more easily attacked by
one or the other organism group. Thus, fungi are regarded as the
main lignin decomposers (De Boer et al., 2005; Berg and Laskowski,
2006). Cellulose is also believed to be mainly degraded by fungi in
terrestrial habitats, although bacteria can also perform this function
(De Boer et al., 2005). Bacteria are thought to be more competitive
regarding easily available carbon sources, such as simple carbohy-
drates, especially under nitrogen-rich conditions (Bardgett et al.,
1999; Bittman et al., 2005; Moore et al., 2005), but this type of
substrates is also preferentially used by fast-growing opportunistic
fungi, like so-called sugar fungi and yeast.
Although it may be considered unimportant which group of
organism is responsible for the decomposition of plant litter in soil,
* Corresponding author. Tel.: þ46 46 222 42 64; fax: þ46 46 222 41 58.
E-mail address: erland.baath@mbioekol.lu.se (E. Bååth).
and composition of decomposition products (Fischer et al., 2006).
It can also have implications for the food web structure. Different
organisms are predators on fungi and bacteria, and the division of
decomposition into a slow fungal based energy channel and
a faster bacterial based food channel has been suggested to be
a main driver of the composition of the food web (De Ruiter et al.,
1993; Moore et al., 2005). It has also been suggested that large
shifts towards either the fungal or the bacterial pathway would
result in unstable conditions (Moore et al., 2005). Apart from the
composition of the substrate (Rousk and Bååth, 2007a), factors
such as soil moisture and temperature, nutrient availability and
extent of fragmentation have also been suggested to affect the
balance between fungal and bacterial decomposition (Paul and
Clark, 1996; Jensen et al., 2003; De Boer et al., 2005; De Vries et al.,
2006; Pietikäinen et al., 2005). However, we have little knowledge
regarding which factors actually determine the division between
fungal and bacterial decomposition. This is partly due to a lack of
analysis techniques. The effect of different environmental factors
on fungi and bacteria is usually studied by measuring changes in
biomass using techniques that can differentiate between these two
groups of organisms. This often requires studies over a long period

0038-0717/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2008.05.011
 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343
of time to be able to detect biomass changes. Furthermore, in-
creased biomass in one group due to favourable conditions can be
counteracted by increased predation, confounding the in-
terpretation of the results. Studying the changes in fungal and
bacterial predator biomass as indications of changes in fungal and
bacterial growth has been suggested as a way of overcoming this
problem (Bjørnlund and Christensen, 2005; Ferris and Bongers,
2006). This has, however, not always been successful (Bouwman
et al., 2005).
A more direct way of studying differential effects on fungi and
bacteria is to study growth. This would also allow short-term
measurements. Although it is possible to estimate bacterial growth
in soil using the thymidine (TdR) or leucine (Leu) incorporation
technique (Bååth, 1992, 1994), a similar method for measuring
fungal growth rates has been lacking. A method initially developed
to measure fungal growth rates on detritus in aquatic habitats
(Newell and Fallon, 1991) was, however, adapted for use in soil
(Bååth, 2001). The method is based on the incorporation of radio-
actively labelled acetate into the fungal-specific substance ergos-
terol (Ac-in-Erg). Recently these methods indicating bacterial and
fungal growth were used to compare effects of adding different
plant materials (Rousk and Bååth, 2007a), as well as for studying
competition between fungi and bacteria in soil (Rousk et al., 2008).
In the present study we compared the response of soil fungal
and bacterial growth to the addition of simple and more complex
organic substrates, added separately or in combination with ni-
trogen. This was performed using the above mentioned techniques.
The total microbial activity was estimated by soil respiration for
comparison. Fungal growth rates were also compared with biomass
estimated using ergosterol or the phospholipid fatty acid 18:2u6,9,
while bacterial biomass was estimated as a sum of bacterial
phospholipid fatty acids. The following main questions were
studied. First, are bacteria more favoured than fungi by easily
available carbon (glucose) compared to a more complex one (cel-
lulose) and vice versa? Second, are bacteria more favoured than
fungi by more available nitrogen (glucose þ nitrogen or gelatine)
compared with carbon-rich conditions (only glucose)? Third, to
what extent could the growth of the microorganisms after adding
different substrates be explained by which nutrient (carbon or ni-
trogen) was limiting microbial growth? We also wanted to eluci-
date possible competition between these organism groups and
investigate whether the activity of one group facilitated the growth
of the other, indicating synergistic interactions. Finally, we studied
the extent to which the total activity (respiration rate) could be
explained by the fungal or bacterial growth rates or a combination
of both. Earlier results indicate that respiration rates in soil are not
always correlated to fungal and bacterial growth (Rajapaksha et al.,
2004; Rousk and Bååth, 2007a).
2. Materials and methods
2.1. Soil and experimental setup
A garden soil (pH(H2O) 6.5, 15.4% organic matter, 20% soil
moisture) was sieved (2 mm mesh size). The soil had a fungal:bac-
terial biomass index (using fungal and bacterial indicator PLFAs) of
0.035, which is at the lower range of soils studied by Frostegård and
Bååth (1996). The soil was divided into 150 g portions, which were
placed in plastic bottles with lids. Seven amendment treatments
(duplicate bottles were used for each treatment) were studied:
control (no substrate added, only talcum), nitrogen (N) as ammo-
nium nitrate (0.1 mg N g1 soil), glucose (Glu) (5 mg g1 soil,
equivalent to 2 mg C g1 soil), glucose and nitrogen (Glu þ N)
(amounts as above), cellulose (Cell) (5 mg g1 soil, equivalent to
2 mg C g1 soil), cellulose þ N (Cell þ N) (amounts as above), and
gelatine (Gel) (4.9 mg g1 soil, equivalent to 2 mg C g1 soil and
2335
0.1 mg N g1 soil). Cellulose was added as a powder (fibrous cellu-
lose powder, CF11, Whatman). The substrates were added after
mixing with talcum (4:1) to avoid clumping of the substrate and to
facilitate dispersion in the soil. Only talcum was added to the
control.
The soil was incubated at room temperature (approx. 22  C).
Samples were taken at intervals up to 2 months (3 months for some
measurements). The water content of the soils was regularly
checked and maintained at 20% by adding distilled water if nec-
essary. The pH(H2O) was measured regularly during incubation, but
remained around pH 6.5 in all samples during the entire duration of
the experiment.
2.2. Total microbial activity (soil respiration)
Soil samples of 1 g (2 g of soil were used after 14 days of in-
cubation and onwards) were placed in glass vials, which were
sealed with rubber septa. After 24 h incubation at 22  C the CO2
concentration in the head space was analysed using a gas chro-
matograph. The respiration rate was measured for 54 days.
2.3. Bacterial growth
Bacterial growth was estimated using the thymidine (TdR) and
leucine (Leu) incorporation techniques simultaneously, as de-
scribed by Bååth (1992, 1994), with the modifications introduced by
Bååth et al. (2001). Both TdR and Leu incorporation was used, since
although Leu incorporation is considered more sensitive, theoret-
ically it may be incorporated into fungi. This is not the case with
TdR, since fungi lack the key enzyme needed for TdR incorporation
(Robarts and Zohary, 1993). Briefly, 1 g of soil was mixed with 40 ml
distilled water in 50 ml centrifuge tubes and shaken on a shaker for
15 min at 300 rpm. The slurry was then centrifuged at 1000g, and
1.5 ml of the supernatant was added to 2 ml micro-centrifuge vials.
Five microliter [methyl-3H]-thymidine (TdR, 37 MBq ml1, 925 GBq
mmol1, Amersham; 130 nM final concentration) and 5 ml [U14C]-
leucine (Leu, 1.85 MBq ml1, 11.3 GBq mmol1, Amersham;
520 nmol final concentration) were added and the bacterial com-
munity was allowed to incorporate the labelled substrates for 2 h at
22  C. Washing and radioactive counting were performed as de-
scribed by Bååth et al. (2001). Leu and TdR incorporation rates were
measured for 54 days, while TdR incorporation only was also
measured after 75 and 82 days.
2.4. Fungal growth
Fungal growth was estimated using the acetate-in-ergosterol
(Ac-in-Erg) incorporation technique originally devised by Newell
and Fallon (1991) for aquatic environments and adapted to soil
conditions by Bååth (2001). Briefly, 1.5 ml distilled water, 0.5 ml
1 mM acetate and 30 ml [1,2-14C] acetic acid (7.4 MBq ml1,
2.04 GBq mmol1, Amersham;) were added to 1 g soil in 10 ml test-
tubes. The slurry was incubated for 16 h at 22  C. Measurement of
ergosterol, indicating fungal biomass, and radioactivity in the
ergosterol, indicating fungal growth, were then performed as de-
scribed by Bååth et al. (2001). Fungal growth was measured for 54
days. The ergosterol measurements were lost on the last sampling
occasion due to equipment failure.
2.5. Nutrients limiting bacterial growth
Nutrients limiting bacterial growth in soil (carbon or nitrogen)
were measured at the beginning (after 3 days) and at the end (after
75 and 84 days, also after 89 days for the Glu and Cell treatments) of
the experiment, essentially according to the method of Aldén et al.
(2001) with some modifications. One gram of soil was placed in
2336 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343
50 ml centrifuge tubes and 2 mg glucose-C or 0.1 mg NH4NO3-N
was added. Glucose and NH4NO3 were mixed with talcum (at ratios
of 4:1 and 1:20, respectively). A control to which only talcum was
added was also included. After 48 h incubation at room tempera-
ture the TdR incorporation was measured as described above. TdR
incorporation in the control sample was set to one in the calcula-
tions. Increased bacterial activity after adding glucose indicates C
limitation, while an increase following nitrogen addition indicates
N limitation. Mean values of the two measurements (or three for
Glu and Cell treatments) made at the end of the study are given.
2.6. Phospholipid fatty acid analysis
Phospholipid fatty acids (PLFAs) were analysed during 60 days
of incubation using the method of Frostegård et al. (1993). A bio-
mass index of fungi was then calculated using the PLFA 18:2u6,9
and the sum of bacterial PLFAs was used as an index of bacterial
biomass (Frostegård and Bååth, 1996).
2.7. Statistical analysis
The data were analysed using repeated measurement ANOVA,
where significant differences to the control were tested using
Dunnett’s test. To be able to compare the effect of the different
additives on the various measures of growth and activity, the data
were normalized to the values of the control without substrate
amendments. Since this process gives a ratio, the data were loga-
rithmically transformed to meet the requirements of the ANOVA.
These data were therefore plotted on a logarithmic scale (Fig. 1).
Standard errors were calculated for each sampling time using
separate one-way-ANOVAs.
3. Results
3.1. Respiration rate
Adding glucose (Glu) or glucose and nitrogen (Glu þ N) in-
creased the respiration rate the first day, to 10 and more than 15
times the control value, respectively (Fig. 1A and B). The respiration
rate then decreased, and after 54 days similar values to that in the
control were found. Adding gelatine (Gel) also increased respira-
tion, although it took 4 days to reach peak respiration, which was
followed by a decrease to the control value (Fig. 1E). Adding
cellulose and nitrogen (Cell þ N) resulted in increased respiration
after about 10 days, to peak values of more than 3 times higher
than the control after w20 days, followed by a gradual decline
(Fig. 1D). Adding only cellulose (Cell) gave only a twofold increase
in respiration rate (Fig. 1C). The nitrogen treatment (N) gave sim-
ilar or, at the end of the experiment, slightly lower respiration rates
than in the control (Fig. 1F).
3.2. Bacterial growth
Bacterial growth estimated either with TdR or Leu incorporation
showed similar patterns with the various additions. The N treat-
ment did not differ from the control (Fig. 1F). Adding Glu alone
resulted in about a twofold increase in growth rates, which
remained constant over the whole incubation period (Fig. 1A). TdR
incorporation rates measured after 75 and 82 days still showed
values about twice as high as in the control (data not shown).
Adding Glu þ N (Fig. 1B) or Gel (Fig. 1E) gave similar results, al-
though the initial increase (more than 10-fold) was seen within 2
days for the former treatment, while the peak activity of the latter
was observed after about 10 days. Thereafter, there was a gradual
decrease in incorporation rates to values 4–6 times higher than in
the control after 54 days. Still after 75 and 82 days bacterial growth
was 2–3 times higher than in the control according to TdR in-
corporation measurements (data not shown). Adding Cell þ N
(Fig. 1D) resulted in an increase in bacterial growth, starting after
about 20 days, to levels more than 4 times the control after 54 days,
which remained at the same high level even after 82 days (TdR
incorporation). Cellulose added separately without nitrogen (Cell,
Fig. 1C) gave a much smaller increase in bacterial growth, only
about 2–3 times the control value. This effect was still evident after
54 days, and according to the TdR incorporation rate, after 82 days.
3.3. Fungal growth
The effect of the additives on fungal growth differed consider-
ably from that on the bacterial growth. Initially high values of
fungal growth (up to 4 times the control) were found in the samples
treated with easily available carbon, with and without nitrogen
(Glu, Glu þ N, Gel; Fig. 1A, B and E). However, after 2 to 3 days
fungal growth decreased to the control value, or in the case of the
Glu þ N and Gel treatments below the control value. The cellulose
treatments (Cell and Cell þ N; Fig. 1C and D) initially showed only
slightly higher values than the control. However, after 7 days these
treatments induced even higher fungal growth rates, peaking about
20 to 30 days after the start of the experiment, with the highest
value for the Cell þ N treatment being 4 times the control. Adding N
only did not affect fungal growth (Fig. 1F).
3.4. Correlation between respiration and microbial growth
Since there were no statistical differences between the control
and the N treatment, the mean values of these were used to indicate
the development over time of the different measures in the control
soil. A significant decrease was seen in respiration rate (Fig. 2A,
p < 0.05) and fungal growth (Fig. 2D, p < 0.05), while no differences
were found over time in the bacterial growth measurements, based
on TdR (Fig. 2B) or Leu incorporation rates (Fig. 2C). This indicated
that the respiration rate was more correlated to fungal than bac-
terial growth.
In the treatments with cellulose (with or without N) there also
appeared to be a correlation between respiration rate and fungal
growth. This is most evident in the Cell þ N treatment (Fig. 1D),
where both started to increase, reached peak values, and then de-
creased at the same time. Bacterial growth, on the other hand,
showed the highest values at the end of the incubation period and
was not correlated to the respiration rate. This was also the case
when only cellulose was added (Fig. 1C), although the effect was
smaller. Both treatments with glucose (Glu and Glu þ N) led to the
highest values of both respiration and fungal growth rates during
the first few days after adding the substances (Fig. 1A and B). These
values then decreased to close to the control value, while bacterial
growth, also showing the highest values shortly after the addition
of the substances, decreased slowly with time (Fig. 1B, Glu þ N) or
not at all (Fig. 1A, Glu). The Gel treatment showed peak values of
the different measures at different times: 2 days for fungal growth,
4 days for respiration rate and around 10 days for bacterial growth
(Fig. 1E). In all cases where easily available carbon was added (Glu,
Glu þ N, Gel; Fig. 1A, B and E), respiration and fungal growth rates
thus returned to the level of the control at the end of the experi-
ment, while bacterial growth rates remained 2–6 times higher than
in the control.
3.5. Comparison between Leu and TdR incorporation
Although the TdR and Leu incorporation techniques gave similar
results, it was clear that the extent of the effects differed, e.g., for
the Glu þ N treatment, where Leu incorporation consistently in-
creased more than TdR incorporation over the entire incubation
 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2337

Glu Glu + N

15.8 A Resp *** 15.8 B Resp ***

TdR *** TdR ***
Leu ** Leu ***
10.0 Erg (*) 10.0 Erg
Relative activities

Relative activity
6.3 6.3

4.0 4.0

2.5 2.5

1.6 1.6

1.0 1.0

0.6 0.6
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (days) Time (days)

Cell Cell + N
C Resp * D
TdR *
4.0 Leu (*) 4.0
Erg *
Resp ***
TdR **
Relative activity
Relative activity

2.5 2.5 Leu *

Erg***

1.6 1.6

1.0 1.0

0.6 0.6
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (days) Time (days)

Gel N
15.8 E Resp *** F Resp
TdR *** TdR
Leu *** 4.0 Leu
10.0 Erg Erg
Relative activity

Relative activity

6.3 2.5

4.0
1.6
2.5

1.6 1.0

1.0
0.6

0.6
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (days) Time (days)

Fig. 1. Comparison of the effect of adding different carbon sources, with and without nitrogen, on microbial activity estimated as soil respiration, bacterial activity (TdR and Leu
incorporation) and fungal activity (Ac-in-Erg incorporation). Addition of (A) glucose, (B) glucose þ N, (C) cellulose, (D) cellulose þ N, (E) gelatine and (F) only N. All data were
normalized to the control (No addition) data, which was set to one on each sampling occasion. These relative activities were expressed on a logarithmic scale. Bars indicate the mean
SE obtained from ANOVA for, from left to right, respiration, TdR, Leu and Ac-in-Erg. Levels of significance from the ANOVA are given after the legends: (*)p < 0.1, *p < 0.05, **p < 0.01,
***p < 0.001.
2338 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343

15
3
A B

(DPM x 103 ml-1h-1)

TdR incorporation
(μg CO2 g-1 soil h-1)
Respiration
10
2

1 5

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (days) Time (days)

4 600

incorporation (DPM g-1 16h-1)

C D
(DPM x 103 ml-1 h-1)
Leu incorporation

Ac-in-ergosterol
3
400

200
1

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (days) Time (days)

Fig. 2. The effect of incubation time on microbial activity in soil with no carbon amendment (mean of the control and the N treatment). (A) Soil respiration, (B) bacterial activity
(TdR incorporation), (C) bacterial activity (Leu incorporation) and (D) fungal activity (Ac-in-Erg incorporation). Bars indicate the SE.

period (Fig. 1B). To compare the effects of different additives on indicated by a Leu:TdR incorporation ratio, compared to the control
these two methods of estimating bacterial growth, the ratio of Leu that was greater than 1. An exception to this was observed during
to TdR incorporation was calculated and compared with the control the first week after gelatine addition, where the opposite behaviour
value (Fig. 3). Treatments that led to high bacterial growth (espe- was found, i.e., less effect on Leu than on TdR incorporation.
cially Glu þ N and Gel, but also Glu and Cell þ N in the later part of
the study) all had higher Leu than TdR incorporation rates, as
3.6. Fungal and bacterial biomass

Changes in the fungal biomass were studied using both the PLFA
18:2u6,9 and ergosterol. The ergosterol content was most affected
No addition
by the Cell þ N treatment, starting to increase after about 10 days,
N
to levels 5 times higher than the control after 35 days (Fig. 4A). The
Relative Leu/TdR incorporation ratio

Glu (*)
Glu + N** cellulose treatment without nitrogen (Cell) also increased the er-
Cell gosterol content, although only to levels twice than that of the
2 Cell + N
control. Following Glu þ N addition, the ergosterol content almost
Gel (*)
doubled during the first few days, and then remained stable at
a mean increase of 1.8 times. The Glu and Gel treatments also led to
slightly higher levels of ergosterol (mean values 1.3 times the
control value in both cases, although not significantly different
from the control). The N treatment did not differ from the control.
1 Similar results as for the ergosterol content were found for the PLFA
18:2u6,9 (Fig. 4B), where the Cell þ N treatment increased the ratio
to almost 5 times the control value after about 20 days, while the
Cell treatment resulted in more than 3 times higher values. Fol-
lowing Glu þ N addition, the PLFA 18:2u6,9 content almost doubled
during the first few days, and then remained stable at a mean in-
crease of 1.5 times. The Glu and Gel treatments also led to slightly
0
higher levels of ergosterol (mean values 1.3 and 1.2 times the
0 10 20 30 40 50 60 control value, respectively) although not significantly different
Time (days) from the control. The N treatment did not differ from the control.
Bacterial PLFAs varied much less than the fungal biomass in-
Fig. 3. The effect of adding different carbon sources, with and without nitrogen, on the dicators (Fig. 4C). Following the Gel and Glu þ N additions, bacterial
leucine to thymidine incorporation ratio. All data were normalized to the control
sample as described in Fig. 1. Bars on the control values indicate the SE obtained from
PLFAs increased in the beginning, and stayed at the same levels
ANOVA. Levels of significance from the ANOVA are given after the legends: (*)p < 0.1, during most of the incubation time, resulting in a significant mean
*p < 0.05, **p < 0.01, ***p < 0.001. increase of 1.4 and 1.3 times the control values, respectively.
 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2339

No addition
N
Glu
A No addition 5 B Glu + N**
N Cell***
5 Glu Cell + N***
Relative ergosterol content Glu + N** Gel

Relative PLFA 18:2ω6,9

Cell (*) 4
4 Cell + N***
Gel
3
3

2
2

1 1

0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
Time (days) Time (days)

C No addition
N
Glu*
Glu + N**
Relative bacterial PLFAs

2
Cell
Cell + N
Gel**

0
0 10 20 30 40 50 60
Time (days)

Fig. 4. The effect of adding different carbon sources, with and without nitrogen, on (A) ergosterol content, (B) the fungal PLFA 18:2u6,9 and (C) the sum of bacterial PLFAs. All data
were normalized to the control sample as described in Fig. 1. Bars on the control values indicate the SE obtained from ANOVA. Levels of significance from the ANOVA are given after
the legends: (*)p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001.

Bacterial PLFAs in the Glu treatment also increased significantly

(mean increase 1.3 times the control), while the other treatments
were not significantly different from the control. However, the
highest values for the bacterial PLFAs in the Cell and Cell þ N
treatments were found at the two last sampling occasions.

3.7. Nutrient limitation

Nutrients limiting bacterial growth were identified at the be-

ginning (short-term, 3 days) and at the end of the experiment
(long-term, 75–89 days, Fig. 5). Bacterial growth in the control
sample was limited by a lack of carbon throughout the experiment,
as evidenced by the significant increase in bacterial activity upon
adding glucose, both at the beginning and at the end of the study,
while adding nitrogen had no effect. In all treatments where N was
added (N, Glu þ N, Cell þ N and Gel) the bacterial community was
also limited by a lack of carbon, with increased bacterial growth
after the addition of glucose, but adding extra nitrogen had no ef-
fect (the latter was studied only at the beginning of the experiment Fig. 5. Factors limiting bacterial growth (TdR incorporation) in the short-term (3 days)
for treatments N and Glu þ N). The Glu treatment altered the bac- and in the long-term (2.5–3 months) in an experiment adding different carbon sources,
with and without nitrogen, to the soil. C was added as glucose or N as ammonium nitrate
terial community from being limited by a lack of carbon to being to soil and bacterial growth after 48 h was compared with a control without amend-
nitrogen limited at the beginning of the study. However, at the end ments. All data were normalized to the control, which was set to one on each sampling
of the experiment neither carbon nor nitrogen application occasion. Statistical differences (*p < 0.05) compared with the control are indicated.
2340 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343
increased bacterial growth (investigated 3 times between days 75
and 89). The same results were found for the Cell treatment.
4. Discussion
4.1. Substrate effects on microbial activity
The main results, that cellulose addition mainly favoured fungal
growth (seen both as an increase in Ac-in-erg incorporation and in
biomass proxies) and that bacteria was initially more favoured by
adding glucose and gelatine, are in accordance with earlier results
using biomass estimations. For example, Van der Wal et al. (2006)
found that the ergosterol content increased more after adding
cellulose to soil compared to adding glucose, while bacterial
numbers were highest after adding glucose. The direct effect on
bacterial, but also fungal growth, after adding easily available sugar
was also reported by Engelking et al. (2007) using amino sugars as
proxies of fungal and bacterial biomass. They also showed that the
fungal biomass increase was delayed after adding cellulose com-
pared with adding easily available sugar.
Also in accordance with earlier studies (see e.g., Dilly, 2004),
respiration rates showed rapid increase after adding an easily
available substrate (glucose), while adding a more complex sub-
strate (cellulose) led to a longer lag time and a smaller increase in
respiration rate (Fig. 1). Cellulose must first be degraded to glucose
monomers, before it can be utilized by the microorganisms.
Cellulolytic enzymes therefore have to be induced, explaining the
lag period. A lag period was also observed upon adding gelatine,
although only a few days, before peak respiration was reached. This
is probably due to the protein first having to be degraded into
peptides and amino acids before the microorganisms can utilize it
for growth and respiration. However, protein degradation is faster
than cellulose degradation. The difference in lag time following the
addition of glucose and gelatine was also seen in the development
of bacterial growth (Fig. 1B and E).
Our first main question, if bacteria would be favoured by adding
an easily available carbon source was only partly confirmed (Fig. 1),
since an increase in fungal growth was also observed during the
first few days. There was also an increase in ergosterol and PLFA
18:2u6,9 content during the first few days following these treat-
ments, indicating fungal growth (Fig. 4A and B). An increase in
ergosterol content 2 weeks after adding glucose to arable soil was
also found by Van der Wal et al. (2006), while Engelking et al.
(2007) found increased ergosterol content 5 days after adding
sucrose to soil. It is possible that this initial growth of fungi is due to
the growth of fast-growing, opportunistic yeasts that are tolerant to
the changes in osmotic conditions induced by the substrate addi-
tions. Adding high amounts of glucose to soil has been found to
drastically increase the number of yeast cells (Bååth et al., 1978),
and yeasts are also favoured by the high concentration of glucose
added in the Substrate Induced Respiration technique used for
biomass estimation (Nordgren, personal communication). In-
creased abundance of the yeast Cryptococcus was also found after
adding glucose to soil (Van der Wal et al., 2006). However, other
fast-growing opportunistic fungi could also be involved in this
initial phase. These fungi are apparently rapidly outcompeted by
the bacteria in these cases, since the fungal growth rates returned
to that of the control within 3 days.
4.2. Antagonistic and synergistic effects
Except for the first few days, easily available carbon appeared to
favour bacterial growth, while fungal growth rates were similar to,
or lower than in, the control (Fig. 1). We cannot rule out the pos-
sibility that this is a technical problem, in that a high bacterial
activity will decrease the amount of acetate available for the fungi
to incorporate into ergosterol during the incubation period of the
Ac-in-erg methodology. It could, however, indicate that bacteria
had antagonistic effects on the fungi. Such an antagonistic re-
lationship has recently been reported for aquatic habitats, where
the growth of bacteria appeared to suppress fungal growth (Gulis
and Suberkropp, 2003; Mille-Lindblom and Tranvik, 2003; Mille-
Lindblom et al., 2006; Romanı́ et al., 2006). It has also been reported
that there was a decrease in bacterial activity due to the addition of
heavy metals (Rajapaksha et al., 2004; Tobor-Kap1on et al., 2005),
NaCl addition (Tobor-Kap1on et al., 2005) or bacterial antibiotics
(Hu and van Bruggen, 1997; Rousk et al., 2008) resulting in in-
creased fungal growth, also indicating antagonistic effects of bac-
teria. Such effects could be due to competition for substrate
(exploitation competition) between these two organism groups, or
to the production of fungal antibiotics or chitinolytic enzymes by
the bacteria (interference competition), although a combination of
these two mechanisms probably is normal. Both these mechanisms
have previously been suggested to be the cause of bacterial an-
tagonism towards fungi (De Boer et al., 1998, 2003; Møller et al.,
1999). Since the antagonistic effect was found after adding a sur-
plus of substrate, it is more likely that interference competition
explains most of the effect. It is interesting in this context that De
Boer et al. (2007) recently reported that non-antagonistic bacteria,
when present in a community, suppressed fungal growth indicating
that antagonism of bacteria towards fungi could be more common
than earlier anticipated. However, Rousk et al. (2008) found in-
dication of antagonistic effects of bacteria also in non-amended
soils, indicating that exploitation competition may also be of
importance.
Fungi have also been reported to have antagonistic effects on
bacteria in soil and water (see e.g., Olsson et al., 1996; Mille-
Lindblom et al., 2006). However, in our study increased fungal
growth after cellulose addition had synergistic effects, in that
bacterial growth started to increase only after fungal growth had
increased (Fig. 1C and D). Fungi are the main cellulose decomposers
in soil (De Boer et al., 2005). However, since the initial degradation
of cellulose to monomers is extracellular, it is possible that bacteria
could act as scavengers and assimilate and grow on these mono-
mers or on organic molecules released by fungi during growth. This
explanation was also suggested in a study where fungal growth on
recalcitrant Phragmites culms enhanced bacterial growth (Romanı́
et al., 2006). However, that the increase in bacterial growth was due
to cellulolytic bacteria could, of course, not be ruled out.
4.3. Effect of nitrogen
The effect of adding nitrogen appeared to depend on the carbon
source. Adding nitrogen together with glucose increased bacterial
growth more than adding glucose alone, while fungal growth was
positively affected by adding nitrogen together with cellulose. It
appears that the carbon source largely determines which group of
microorganisms is favoured. In both cases adding only the carbon
source will change the soil from being limited by a lack of carbon to
being limited by a lack of nitrogen (Fig. 1A and B for bacteria and C
and D for fungi). Adding the limiting nutrient would then alleviate
the limitation, resulting in greater growth of the organism group
most suited to the particular carbon source. Similar results were
found by Rousk and Bååth (2007a) when studying fungal and
bacterial growth after adding alfalfa or straw to soil. Straw
amendment favoured fungal growth and adding nitrogen to straw
amended soils increased fungal growth even more.
4.4. Respiration and microbial growth
So far, the only way of differentiating between CO2 emanating
from bacteria and fungi has been to add antibiotics specific to one of
 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343
these two groups and determine the extent of the decrease in
respiration. This is the basic principle behind the Selective In-
hibition technique for estimating fungal and bacterial biomass
(Anderson and Domsch, 1973; Bailey et al., 2003). However, this
requires the addition of large concentrations of antibiotics and
triggering of the respiration rate by adding glucose. Although we
could not directly measure the contribution of fungi and bacteria to
CO2 production, the often very close correlation between fungal
growth and respiration rates suggests that fungi were the main
contributors to soil respiration. This is most clearly seen after
adding cellulose alone or with nitrogen (Fig. 1C and D). Initially high
respiration rates, which decreased rapidly within 3 days of adding
an easily available carbon source, were also better correlated to the
initial increase in fungal growth than to bacterial growth (Fig. 1A, B
and E). This was even more evident at the end of the incubation
period, when respiration rates and fungal growth had returned to
control values, while bacterial growth was still high. To be able
to accurately determine the contribution of these organism groups
to CO2 production in soil we need estimates of fungal and bacterial
growth efficiency. However, it is difficult to determine growth
efficiency in soil. The possibility of combining measurements of
fungal and bacterial growth, and selective inhibition, isolating the
activity of one of the groups, offers the possibility of such de-
terminations in a similar way to that used for determining bacterial
growth efficiency in aquatic habitats (Del Giorgio and Cole, 1998).
The increased bacterial growth resulting from the addition of
easily available substrates persisted for at least 3 months, long after
the effects on respiration and fungal growth had disappeared. This
persistent increase in TdR and Leu incorporation rates over long
periods was also reported by Vinten et al. (2002) after adding
glucose and by Rousk and Bååth (2007a) after adding alfalfa. Also,
chloroform fumigation and recolonization resulted in high values
of these measures which still were evident 8 months after the
initial perturbation (Bååth, unpublished). Assuming that this is not
an artefact of the methodology, this implies that bacterial growth is
fuelled in a different way from fungal growth, and that the resulting
respiration rate hitherto used for estimating microbial activity in
soil underestimates bacterial growth. The mechanism behind this,
i.e., the use of intracellular storage products or extracellular de-
composition products, or predation stimulating bacterial pro-
duction, remains to be determined. However, this clearly illustrates
the increased information on the soil system obtained by com-
bining measurements of the growth and activities of the different
organism groups.
4.5. Microbial growth and biomass
There appeared to be a good correlation between the changes in
fungal biomass, measured both with ergosterol and the PLFA
18:2u6,9, and fungal growth, measured using Ac-in-erg in-
corporation. This has earlier been found after adding plant material
to soil (Rousk and Bååth, 2007b). However, bacterial biomass (es-
timated using bacterial PLFAs) was much more static despite large
variations in bacterial growth rates, even if the main results (largest
increase in both bacterial PLFAs and growth after adding Glu þ N
and Gel, followed by adding Glu only) was similar for both mea-
sures. Also, the prolonged time with increased bacterial growth
rates even after 3 months was not reflected in a continuous increase
in bacterial PLFAs. Similar discrepancies were earlier reported after
adding plant substrates to soil, where adding alfalfa meal increased
bacterial growth rates even after 2 months incubation with no
concomitant increase in bacterial PLFAs (Rousk and Bååth, 2007a).
The authors discussed possible explanations for this, e.g., bacterial
PLFAs including large amounts of inactive biomass, different turn-
overs of bacterial and fungal PLFAs, and the extent of predation
differing between fungi and bacteria. Bacterial predators are known
2341
to increase rapidly in soil treated to increase bacterial growth
(Saetre and Stark, 2005; Christensen et al., 2007). If the latter ex-
planation is valid, small or unaltered changes in bacterial biomass
could well be found, even if bacterial growth and production is
increased.
4.6. Comparison between Leu and TdR incorporation
The Leu:TdR incorporation ratio has been found to be positively
correlated to bacterial activity in aquatic ecosystems (Servais,
1992). In a recent study, Longnecker et al. (2006) also found that
higher Leu:TdR incorporation ratios were associated with the more
active members of bacterioplankton, that is, those with high nucleic
acid content. Similar results were found in the present study, in that
the treatments leading to the highest increase in bacterial activities
exhibited the highest Leu:TdR incorporation ratios (Fig. 3). Vinten
et al. (2002) added glucose to a soil and found an eightfold increase
in TdR incorporation rates, while Leu incorporation increased 16
times compared with the unamended control. Increasing Leu:TdR
incorporation ratios were also found after substrate (alfalfa and
straw) additions (Rousk and Bååth, 2007a). Thus, an increased
Leu:TdR incorporation ratio with higher bacterial growth rates
appears to be a consistent finding in both aquatic and soil habitats.
Longnecker et al. (2006) suggested that the increased Leu:TdR in-
corporation ratio was due to larger, fast-growing cells containing
more protein, since the protein content of a cell is correlated to the
cell volume (Simon and Azam, 1989). This suggests that the Leu
incorporation technique would be more sensitive in measuring the
effects of additives that increase bacterial growth than the TdR
incorporation technique.
However, during the first week after adding gelatine, the Leu:TdR
incorporation ratio was low despite high bacterial growth rates
(Figs. 3 and 1E). This could be due to an isotope dilution effect
resulting from increased amino acid concentrations in the soil
arising from the degradation of gelatine during this period. Although
earlier results have shown isotope dilution for Leu incorporation to
be rather constant in soil (Bååth,1998), this is apparently not the case
when large amounts of proteins are added. Thus, when adding
a protein-containing substrate to soil, the interpretation of Leu in-
corporation rate as growth may result in underestimation of the true
growth rates. Combining the Leu and TdR incorporation techniques
is a good way of elucidating such confounding effects.
4.7. Nutrient limitation
Bacterial growth was limited by a lack of carbon throughout the
incubation period in the control soil (Fig. 5), as was the case in the
treatments where nitrogen was added. However, we expected that
adding only large amounts of carbon (Glu and Cell treatments)
would cause nitrogen limitation of the bacteria. This was the case in
the short-term for glucose addition (cellulose addition not in-
vestigated), but after 3 months of incubation the addition of neither
glucose-C nor nitrogen affected bacterial growth. One explanation
of this may be that both carbon and nitrogen were close to limiting
bacterial growth, and adding both simultaneously would be
necessary to increase growth. This situation has been observed
previously in some soils (Demoling et al., 2007). However, the
bacteria may have been limited by a lack of nitrogen, but the in-
cubation time used (48 h) too short to observe a growth response.
Schimel and Weintraub (2003) modelled the effect of adding car-
bon when microbial growth was carbon limited, and nitrogen when
it was nitrogen limited. In the former case growth rates increased
rapidly, but in the latter the increase in microbial biomass upon
adding nitrogen was also controlled by the flow of carbon from soil
organic matter. This is most easily explained in the situation with
cellulose. Although this may induce nitrogen limitation of bacterial
2342 S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343
growth, adding nitrogen would not result in an immediate increase
in bacterial growth. Exoenzymes produced by the fungi must first
degrade the cellulose to products available to the bacteria. It is less
evident for the glucose treatment. The glucose added may, how-
ever, have been transformed into other substances that were not
immediately available during the 3-month incubation period, for
example to storage products such as poly-beta-hydroxybutyrate.
5. Conclusions
In conclusion, we have shown that both bacteria and fungi were
favoured by an easily available carbon source, although with a dif-
ferent time frame, while fungi were initially more favoured by
growth on the complex substrate cellulose. Nitrogen addition did
not favour bacterial growth, but allowed the group that was already
favoured by the complexity of the substrate to grow better, i.e.,
adding nitrogen together with cellulose favoured fungi, while
adding nitrogen together with glucose favoured bacteria. Fungal
growth on cellulose facilitated the growth of bacteria, while in-
creased growth of bacteria had an antagonistic effect on fungal
growth. Finally, fungal growth appeared to be better correlated to
respiration rates than bacterial growth, indicating that the former
group was more important for decomposition. The use of the
measures indicating bacterial growth and fungal growth allowed
rapid and sensitive estimations of changes in growth, in both in-
creasing and decreasing phases. Also, the possibility of combining
these methods, applying conversion factors to calculate biomass
production (e.g., Rousk and Bååth, 2007b), with respiration mea-
surements provides the possibility of estimating soil microorgan-
ism efficiency.
Acknowledgements
This study was supported by a grant from the Swedish Research
Council to E.B.
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