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Soil Biology & Biochemistry 40 (2008) 2334–2343

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Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lb io

Antagonistic and synergistic effects of fungal and bacterial growth in soil afteradding 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

Article history:Received 8 January 2008Received in revised form 29 April 2008Accepted 13 May 2008Available online 11 June 2008

Keywords:FungiBacteriaThymidineLeucine14C acetateErgosterol

* Corresponding author. Tel.: þ46 46 222 42 64; faE-mail address: erland.baath@mbioekol.lu.se (E. B

0038-0717/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.soilbio.2008.05.011

a b s t r a c t

The effect of adding easily available and more complex carbon sources, with and without nitrogen, onfungal and bacterial growth and activity in soil were studied in the laboratory. Total microbial activitywas estimated by measuring respiration, fungal growth with the acetate-in-ergosterol incorporationtechnique and bacterial growth with the thymidine and leucine incorporation techniques. The substrateadditions consisted of glucose and cellulose, with and without nitrogen (as ammonium nitrate), andgelatine. The microbial development was followed over a 2-month period. The respiration rate increasedwithin a few days after adding glucose, with and without nitrogen, and gelatine, initially by more than 10times, but after 2 months no differences were seen compared with the control. Bacterial growth esti-mated with the thymidine and leucine incorporation techniques gave similar results. Adding glucosewith nitrogen, or gelatine, increased bacterial growth within a few days up to 10 times, but even after 2months of incubation bacterial growth rates were still about 5 times higher than in the control. Addingonly glucose increased bacterial growth rates by about twice over the whole incubation period. Fungalgrowth rates especially increased after adding cellulose and nitrogen, although a minor increase wasfound after adding cellulose alone. Fungal growth rates started to increase after 10 days of incubationwith cellulose. There were indications of synergistic effects in that bacterial growth increased after thefungi 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.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Bacteria and fungi are the dominating groups of organismsfound 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 byone or the other organism group. Thus, fungi are regarded as themain lignin decomposers (De Boer et al., 2005; Berg and Laskowski,2006). Cellulose is also believed to be mainly degraded by fungi interrestrial habitats, although bacteria can also perform this function(De Boer et al., 2005). Bacteria are thought to be more competitiveregarding 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 ofsubstrates is also preferentially used by fast-growing opportunisticfungi, like so-called sugar fungi and yeast.

Although it may be considered unimportant which group oforganism is responsible for the decomposition of plant litter in soil,

x: þ46 46 222 41 58.ååth).

All rights reserved.

bacterial or fungal decomposition can result in different amountsand composition of decomposition products (Fischer et al., 2006).It can also have implications for the food web structure. Differentorganisms are predators on fungi and bacteria, and the division ofdecomposition into a slow fungal based energy channel anda faster bacterial based food channel has been suggested to bea main driver of the composition of the food web (De Ruiter et al.,1993; Moore et al., 2005). It has also been suggested that largeshifts towards either the fungal or the bacterial pathway wouldresult in unstable conditions (Moore et al., 2005). Apart from thecomposition of the substrate (Rousk and Bååth, 2007a), factorssuch as soil moisture and temperature, nutrient availability andextent of fragmentation have also been suggested to affect thebalance between fungal and bacterial decomposition (Paul andClark, 1996; Jensen et al., 2003; De Boer et al., 2005; De Vries et al.,2006; Pietikainen et al., 2005). However, we have little knowledgeregarding which factors actually determine the division betweenfungal and bacterial decomposition. This is partly due to a lack ofanalysis techniques. The effect of different environmental factorson fungi and bacteria is usually studied by measuring changes inbiomass using techniques that can differentiate between these twogroups of organisms. This often requires studies over a long period

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2335

of time to be able to detect biomass changes. Furthermore, in-creased biomass in one group due to favourable conditions can becounteracted by increased predation, confounding the in-terpretation of the results. Studying the changes in fungal andbacterial predator biomass as indications of changes in fungal andbacterial growth has been suggested as a way of overcoming thisproblem (Bjørnlund and Christensen, 2005; Ferris and Bongers,2006). This has, however, not always been successful (Bouwmanet al., 2005).

A more direct way of studying differential effects on fungi andbacteria is to study growth. This would also allow short-termmeasurements. Although it is possible to estimate bacterial growthin soil using the thymidine (TdR) or leucine (Leu) incorporationtechnique (Bååth, 1992, 1994), a similar method for measuringfungal growth rates has been lacking. A method initially developedto 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 andfungal growth were used to compare effects of adding differentplant materials (Rousk and Bååth, 2007a), as well as for studyingcompetition between fungi and bacteria in soil (Rousk et al., 2008).

In the present study we compared the response of soil fungaland bacterial growth to the addition of simple and more complexorganic 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 forcomparison. Fungal growth rates were also compared with biomassestimated using ergosterol or the phospholipid fatty acid 18:2u6,9,while bacterial biomass was estimated as a sum of bacterialphospholipid fatty acids. The following main questions werestudied. First, are bacteria more favoured than fungi by easilyavailable carbon (glucose) compared to a more complex one (cel-lulose) and vice versa? Second, are bacteria more favoured thanfungi by more available nitrogen (glucoseþ nitrogen or gelatine)compared with carbon-rich conditions (only glucose)? Third, towhat extent could the growth of the microorganisms after addingdifferent 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 andinvestigate whether the activity of one group facilitated the growthof the other, indicating synergistic interactions. Finally, we studiedthe extent to which the total activity (respiration rate) could beexplained by the fungal or bacterial growth rates or a combinationof both. Earlier results indicate that respiration rates in soil are notalways 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% soilmoisture) was sieved (2 mm mesh size). The soil had a fungal:bac-terial biomass index (using fungal and bacterial indicator PLFAs) of0.035, which is at the lower range of soils studied by Frostegård andBååth (1996). The soil was divided into 150 g portions, which wereplaced 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 g�1 soil), glucose (Glu) (5 mg g�1 soil,equivalent to 2 mg C g�1 soil), glucose and nitrogen (GluþN)(amounts as above), cellulose (Cell) (5 mg g�1 soil, equivalent to2 mg C g�1 soil), celluloseþN (CellþN) (amounts as above), andgelatine (Gel) (4.9 mg g�1 soil, equivalent to 2 mg C g�1 soil and

0.1 mg N g�1 soil). Cellulose was added as a powder (fibrous cellu-lose powder, CF11, Whatman). The substrates were added aftermixing with talcum (4:1) to avoid clumping of the substrate and tofacilitate dispersion in the soil. Only talcum was added to thecontrol.

The soil was incubated at room temperature (approx. 22 �C).Samples were taken at intervals up to 2 months (3 months for somemeasurements). The water content of the soils was regularlychecked and maintained at 20% by adding distilled water if nec-essary. The pH(H2O) was measured regularly during incubation, butremained around pH 6.5 in all samples during the entire duration ofthe 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 weresealed 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) andleucine (Leu) incorporation techniques simultaneously, as de-scribed by Bååth (1992, 1994), with the modifications introduced byBååth et al. (2001). Both TdR and Leu incorporation was used, sincealthough Leu incorporation is considered more sensitive, theoret-ically it may be incorporated into fungi. This is not the case withTdR, since fungi lack the key enzyme needed for TdR incorporation(Robarts and Zohary, 1993). Briefly, 1 g of soil was mixed with 40 mldistilled water in 50 ml centrifuge tubes and shaken on a shaker for15 min at 300 rpm. The slurry was then centrifuged at 1000�g, and1.5 ml of the supernatant was added to 2 ml micro-centrifuge vials.Five microliter [methyl-3H]-thymidine (TdR, 37 MBq ml�1, 925 GBqmmol�1, Amersham; 130 nM final concentration) and 5 ml [U14C]-leucine (Leu, 1.85 MBq ml�1, 11.3 GBq mmol�1, Amersham;520 nmol final concentration) were added and the bacterial com-munity was allowed to incorporate the labelled substrates for 2 h at22 �C. Washing and radioactive counting were performed as de-scribed by Bååth et al. (2001). Leu and TdR incorporation rates weremeasured for 54 days, while TdR incorporation only was alsomeasured 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 Newelland Fallon (1991) for aquatic environments and adapted to soilconditions by Bååth (2001). Briefly, 1.5 ml distilled water, 0.5 ml1 mM acetate and 30 ml [1,2-14C] acetic acid (7.4 MBq ml�1,2.04 GBq mmol�1, Amersham;) were added to 1 g soil in 10 ml test-tubes. The slurry was incubated for 16 h at 22 �C. Measurement ofergosterol, indicating fungal biomass, and radioactivity in theergosterol, indicating fungal growth, were then performed as de-scribed by Bååth et al. (2001). Fungal growth was measured for 54days. The ergosterol measurements were lost on the last samplingoccasion 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 (after75 and 84 days, also after 89 days for the Glu and Cell treatments) ofthe experiment, essentially according to the method of Alden et al.(2001) with some modifications. One gram of soil was placed in

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–23432336

50 ml centrifuge tubes and 2 mg glucose-C or 0.1 mg NH4NO3-Nwas added. Glucose and NH4NO3 were mixed with talcum (at ratiosof 4:1 and 1:20, respectively). A control to which only talcum wasadded was also included. After 48 h incubation at room tempera-ture the TdR incorporation was measured as described above. TdRincorporation in the control sample was set to one in the calcula-tions. Increased bacterial activity after adding glucose indicates Climitation, while an increase following nitrogen addition indicatesN limitation. Mean values of the two measurements (or three forGlu 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 daysof incubation using the method of Frostegård et al. (1993). A bio-mass index of fungi was then calculated using the PLFA 18:2u6,9and the sum of bacterial PLFAs was used as an index of bacterialbiomass (Frostegård and Bååth, 1996).

2.7. Statistical analysis

The data were analysed using repeated measurement ANOVA,where significant differences to the control were tested usingDunnett’s test. To be able to compare the effect of the differentadditives on the various measures of growth and activity, the datawere normalized to the values of the control without substrateamendments. 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 usingseparate one-way-ANOVAs.

3. Results

3.1. Respiration rate

Adding glucose (Glu) or glucose and nitrogen (GluþN) in-creased the respiration rate the first day, to 10 and more than 15times the control value, respectively (Fig. 1A and B). The respirationrate then decreased, and after 54 days similar values to that in thecontrol were found. Adding gelatine (Gel) also increased respira-tion, although it took 4 days to reach peak respiration, which wasfollowed by a decrease to the control value (Fig. 1E). Addingcellulose and nitrogen (CellþN) resulted in increased respirationafter about 10 days, to peak values of more than 3 times higherthan the control after w20 days, followed by a gradual decline(Fig. 1D). Adding only cellulose (Cell) gave only a twofold increasein respiration rate (Fig. 1C). The nitrogen treatment (N) gave sim-ilar or, at the end of the experiment, slightly lower respiration ratesthan in the control (Fig. 1F).

3.2. Bacterial growth

Bacterial growth estimated either with TdR or Leu incorporationshowed similar patterns with the various additions. The N treat-ment did not differ from the control (Fig. 1F). Adding Glu aloneresulted in about a twofold increase in growth rates, whichremained constant over the whole incubation period (Fig. 1A). TdRincorporation rates measured after 75 and 82 days still showedvalues about twice as high as in the control (data not shown).Adding GluþN (Fig. 1B) or Gel (Fig. 1E) gave similar results, al-though the initial increase (more than 10-fold) was seen within 2days for the former treatment, while the peak activity of the latterwas observed after about 10 days. Thereafter, there was a gradualdecrease in incorporation rates to values 4–6 times higher than inthe 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 CellþN(Fig. 1D) resulted in an increase in bacterial growth, starting afterabout 20 days, to levels more than 4 times the control after 54 days,which remained at the same high level even after 82 days (TdRincorporation). Cellulose added separately without nitrogen (Cell,Fig. 1C) gave a much smaller increase in bacterial growth, onlyabout 2–3 times the control value. This effect was still evident after54 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 offungal growth (up to 4 times the control) were found in the samplestreated with easily available carbon, with and without nitrogen(Glu, GluþN, Gel; Fig. 1A, B and E). However, after 2 to 3 daysfungal growth decreased to the control value, or in the case of theGluþN and Gel treatments below the control value. The cellulosetreatments (Cell and CellþN; Fig. 1C and D) initially showed onlyslightly higher values than the control. However, after 7 days thesetreatments induced even higher fungal growth rates, peaking about20 to 30 days after the start of the experiment, with the highestvalue for the CellþN treatment being 4 times the control. Adding Nonly did not affect fungal growth (Fig. 1F).

3.4. Correlation between respiration and microbial growth

Since there were no statistical differences between the controland the N treatment, the mean values of these were used to indicatethe development over time of the different measures in the controlsoil. A significant decrease was seen in respiration rate (Fig. 2A,p< 0.05) and fungal growth (Fig. 2D, p< 0.05), while no differenceswere found over time in the bacterial growth measurements, basedon TdR (Fig. 2B) or Leu incorporation rates (Fig. 2C). This indicatedthat the respiration rate was more correlated to fungal than bac-terial growth.

In the treatments with cellulose (with or without N) there alsoappeared to be a correlation between respiration rate and fungalgrowth. This is most evident in the CellþN 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 andwas not correlated to the respiration rate. This was also the casewhen only cellulose was added (Fig. 1C), although the effect wassmaller. Both treatments with glucose (Glu and GluþN) led to thehighest values of both respiration and fungal growth rates duringthe first few days after adding the substances (Fig. 1A and B). Thesevalues then decreased to close to the control value, while bacterialgrowth, also showing the highest values shortly after the additionof the substances, decreased slowly with time (Fig. 1B, GluþN) ornot at all (Fig. 1A, Glu). The Gel treatment showed peak values ofthe 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,GluþN, Gel; Fig. 1A, B and E), respiration and fungal growth ratesthus returned to the level of the control at the end of the experi-ment, while bacterial growth rates remained 2–6 times higher thanin the control.

3.5. Comparison between Leu and TdR incorporation

Although the TdR and Leu incorporation techniques gave similarresults, it was clear that the extent of the effects differed, e.g., forthe GluþN treatment, where Leu incorporation consistently in-creased more than TdR incorporation over the entire incubation

Glu

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0.6

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Resp ***TdR ***Leu ***Erg

RespTdRLeuErg

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 Leuincorporation) and fungal activity (Ac-in-Erg incorporation). Addition of (A) glucose, (B) glucoseþN, (C) cellulose, (D) celluloseþN, (E) gelatine and (F) only N. All data werenormalized 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 meanSE 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.

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2337

0

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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.

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–23432338

period (Fig. 1B). To compare the effects of different additives onthese two methods of estimating bacterial growth, the ratio of Leuto TdR incorporation was calculated and compared with the controlvalue (Fig. 3). Treatments that led to high bacterial growth (espe-cially GluþN and Gel, but also Glu and CellþN in the later part ofthe study) all had higher Leu than TdR incorporation rates, as

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No additionNGlu (*)Glu + N**CellCell + NGel (*)

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Fig. 3. The effect of adding different carbon sources, with and without nitrogen, on theleucine to thymidine incorporation ratio. All data were normalized to the controlsample as described in Fig. 1. Bars on the control values indicate the SE obtained fromANOVA. Levels of significance from the ANOVA are given after the legends: (*)p< 0.1,*p< 0.05, **p< 0.01, ***p< 0.001.

indicated by a Leu:TdR incorporation ratio, compared to the controlthat was greater than 1. An exception to this was observed duringthe first week after gelatine addition, where the opposite behaviourwas found, i.e., less effect on Leu than on TdR incorporation.

3.6. Fungal and bacterial biomass

Changes in the fungal biomass were studied using both the PLFA18:2u6,9 and ergosterol. The ergosterol content was most affectedby the CellþN treatment, starting to increase after about 10 days,to levels 5 times higher than the control after 35 days (Fig. 4A). Thecellulose treatment without nitrogen (Cell) also increased the er-gosterol content, although only to levels twice than that of thecontrol. Following GluþN addition, the ergosterol content almostdoubled during the first few days, and then remained stable ata mean increase of 1.8 times. The Glu and Gel treatments also led toslightly higher levels of ergosterol (mean values 1.3 times thecontrol value in both cases, although not significantly differentfrom the control). The N treatment did not differ from the control.Similar results as for the ergosterol content were found for the PLFA18:2u6,9 (Fig. 4B), where the CellþN treatment increased the ratioto almost 5 times the control value after about 20 days, while theCell treatment resulted in more than 3 times higher values. Fol-lowing GluþN addition, the PLFA 18:2u6,9 content almost doubledduring 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 slightlyhigher levels of ergosterol (mean values 1.3 and 1.2 times thecontrol value, respectively) although not significantly differentfrom the control. The N treatment did not differ from the control.

Bacterial PLFAs varied much less than the fungal biomass in-dicators (Fig. 4C). Following the Gel and GluþN additions, bacterialPLFAs increased in the beginning, and stayed at the same levelsduring most of the incubation time, resulting in a significant meanincrease of 1.4 and 1.3 times the control values, respectively.

0

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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 datawere 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 afterthe legends: (*)p< 0.1, *p< 0.05, **p< 0.01, ***p< 0.001.

Fig. 5. Factors limiting bacterial growth (TdR incorporation) in the short-term (3 days)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 nitrateto soil and bacterial growth after 48 h was compared with a control without amend-ments. All data were normalized to the control, which was set to one on each samplingoccasion. Statistical differences (*p< 0.05) compared with the control are indicated.

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2339

Bacterial PLFAs in the Glu treatment also increased significantly(mean increase 1.3 times the control), while the other treatmentswere not significantly different from the control. However, thehighest values for the bacterial PLFAs in the Cell and CellþNtreatments 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 controlsample was limited by a lack of carbon throughout the experiment,as evidenced by the significant increase in bacterial activity uponadding glucose, both at the beginning and at the end of the study,while adding nitrogen had no effect. In all treatments where N wasadded (N, GluþN, CellþN and Gel) the bacterial community wasalso limited by a lack of carbon, with increased bacterial growthafter the addition of glucose, but adding extra nitrogen had no ef-fect (the latter was studied only at the beginning of the experimentfor treatments N and GluþN). The Glu treatment altered the bac-terial community from being limited by a lack of carbon to beingnitrogen limited at the beginning of the study. However, at the endof the experiment neither carbon nor nitrogen application

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–23432340

increased bacterial growth (investigated 3 times between days 75and 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 fungalgrowth (seen both as an increase in Ac-in-erg incorporation and inbiomass proxies) and that bacteria was initially more favoured byadding glucose and gelatine, are in accordance with earlier resultsusing biomass estimations. For example, Van der Wal et al. (2006)found that the ergosterol content increased more after addingcellulose to soil compared to adding glucose, while bacterialnumbers were highest after adding glucose. The direct effect onbacterial, but also fungal growth, after adding easily available sugarwas also reported by Engelking et al. (2007) using amino sugars asproxies of fungal and bacterial biomass. They also showed that thefungal 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 easilyavailable substrate (glucose), while adding a more complex sub-strate (cellulose) led to a longer lag time and a smaller increase inrespiration rate (Fig. 1). Cellulose must first be degraded to glucosemonomers, before it can be utilized by the microorganisms.Cellulolytic enzymes therefore have to be induced, explaining thelag period. A lag period was also observed upon adding gelatine,although only a few days, before peak respiration was reached. Thisis probably due to the protein first having to be degraded intopeptides and amino acids before the microorganisms can utilize itfor growth and respiration. However, protein degradation is fasterthan cellulose degradation. The difference in lag time following theaddition of glucose and gelatine was also seen in the developmentof bacterial growth (Fig. 1B and E).

Our first main question, if bacteria would be favoured by addingan easily available carbon source was only partly confirmed (Fig. 1),since an increase in fungal growth was also observed during thefirst few days. There was also an increase in ergosterol and PLFA18:2u6,9 content during the first few days following these treat-ments, indicating fungal growth (Fig. 4A and B). An increase inergosterol content 2 weeks after adding glucose to arable soil wasalso found by Van der Wal et al. (2006), while Engelking et al.(2007) found increased ergosterol content 5 days after addingsucrose to soil. It is possible that this initial growth of fungi is due tothe growth of fast-growing, opportunistic yeasts that are tolerant tothe changes in osmotic conditions induced by the substrate addi-tions. Adding high amounts of glucose to soil has been found todrastically increase the number of yeast cells (Bååth et al., 1978),and yeasts are also favoured by the high concentration of glucoseadded in the Substrate Induced Respiration technique used forbiomass estimation (Nordgren, personal communication). In-creased abundance of the yeast Cryptococcus was also found afteradding glucose to soil (Van der Wal et al., 2006). However, otherfast-growing opportunistic fungi could also be involved in thisinitial phase. These fungi are apparently rapidly outcompeted bythe bacteria in these cases, since the fungal growth rates returnedto that of the control within 3 days.

4.2. Antagonistic and synergistic effects

Except for the first few days, easily available carbon appeared tofavour 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 bacterialactivity will decrease the amount of acetate available for the fungi

to incorporate into ergosterol during the incubation period of theAc-in-erg methodology. It could, however, indicate that bacteriahad antagonistic effects on the fungi. Such an antagonistic re-lationship has recently been reported for aquatic habitats, wherethe growth of bacteria appeared to suppress fungal growth (Gulisand Suberkropp, 2003; Mille-Lindblom and Tranvik, 2003; Mille-Lindblom et al., 2006; Romanı et al., 2006). It has also been reportedthat there was a decrease in bacterial activity due to the addition ofheavy 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, orto the production of fungal antibiotics or chitinolytic enzymes bythe bacteria (interference competition), although a combination ofthese two mechanisms probably is normal. Both these mechanismshave 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 competitionexplains most of the effect. It is interesting in this context that DeBoer et al. (2007) recently reported that non-antagonistic bacteria,when present in a community, suppressed fungal growth indicatingthat antagonism of bacteria towards fungi could be more commonthan earlier anticipated. However, Rousk et al. (2008) found in-dication of antagonistic effects of bacteria also in non-amendedsoils, indicating that exploitation competition may also be ofimportance.

Fungi have also been reported to have antagonistic effects onbacteria in soil and water (see e.g., Olsson et al., 1996; Mille-Lindblom et al., 2006). However, in our study increased fungalgrowth after cellulose addition had synergistic effects, in thatbacterial growth started to increase only after fungal growth hadincreased (Fig. 1C and D). Fungi are the main cellulose decomposersin soil (De Boer et al., 2005). However, since the initial degradationof cellulose to monomers is extracellular, it is possible that bacteriacould act as scavengers and assimilate and grow on these mono-mers or on organic molecules released by fungi during growth. Thisexplanation was also suggested in a study where fungal growth onrecalcitrant Phragmites culms enhanced bacterial growth (Romanıet al., 2006). However, that the increase in bacterial growth was dueto cellulolytic bacteria could, of course, not be ruled out.

4.3. Effect of nitrogen

The effect of adding nitrogen appeared to depend on the carbonsource. Adding nitrogen together with glucose increased bacterialgrowth more than adding glucose alone, while fungal growth waspositively affected by adding nitrogen together with cellulose. Itappears that the carbon source largely determines which group ofmicroorganisms is favoured. In both cases adding only the carbonsource will change the soil from being limited by a lack of carbon tobeing limited by a lack of nitrogen (Fig. 1A and B for bacteria and Cand D for fungi). Adding the limiting nutrient would then alleviatethe limitation, resulting in greater growth of the organism groupmost suited to the particular carbon source. Similar results werefound by Rousk and Bååth (2007a) when studying fungal andbacterial growth after adding alfalfa or straw to soil. Strawamendment favoured fungal growth and adding nitrogen to strawamended soils increased fungal growth even more.

4.4. Respiration and microbial growth

So far, the only way of differentiating between CO2 emanatingfrom bacteria and fungi has been to add antibiotics specific to one of

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2341

these two groups and determine the extent of the decrease inrespiration. 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, thisrequires the addition of large concentrations of antibiotics andtriggering of the respiration rate by adding glucose. Although wecould not directly measure the contribution of fungi and bacteria toCO2 production, the often very close correlation between fungalgrowth and respiration rates suggests that fungi were the maincontributors to soil respiration. This is most clearly seen afteradding cellulose alone or with nitrogen (Fig. 1C and D). Initially highrespiration rates, which decreased rapidly within 3 days of addingan easily available carbon source, were also better correlated to theinitial increase in fungal growth than to bacterial growth (Fig. 1A, Band E). This was even more evident at the end of the incubationperiod, when respiration rates and fungal growth had returned tocontrol values, while bacterial growth was still high. To be ableto accurately determine the contribution of these organism groupsto CO2 production in soil we need estimates of fungal and bacterialgrowth efficiency. However, it is difficult to determine growthefficiency in soil. The possibility of combining measurements offungal and bacterial growth, and selective inhibition, isolating theactivity of one of the groups, offers the possibility of such de-terminations in a similar way to that used for determining bacterialgrowth efficiency in aquatic habitats (Del Giorgio and Cole, 1998).

The increased bacterial growth resulting from the addition ofeasily available substrates persisted for at least 3 months, long afterthe effects on respiration and fungal growth had disappeared. Thispersistent increase in TdR and Leu incorporation rates over longperiods was also reported by Vinten et al. (2002) after addingglucose and by Rousk and Bååth (2007a) after adding alfalfa. Also,chloroform fumigation and recolonization resulted in high valuesof these measures which still were evident 8 months after theinitial perturbation (Bååth, unpublished). Assuming that this is notan artefact of the methodology, this implies that bacterial growth isfuelled in a different way from fungal growth, and that the resultingrespiration rate hitherto used for estimating microbial activity insoil 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 illustratesthe increased information on the soil system obtained by com-bining measurements of the growth and activities of the differentorganism groups.

4.5. Microbial growth and biomass

There appeared to be a good correlation between the changes infungal biomass, measured both with ergosterol and the PLFA18:2u6,9, and fungal growth, measured using Ac-in-erg in-corporation. This has earlier been found after adding plant materialto soil (Rousk and Bååth, 2007b). However, bacterial biomass (es-timated using bacterial PLFAs) was much more static despite largevariations in bacterial growth rates, even if the main results (largestincrease in both bacterial PLFAs and growth after adding GluþNand Gel, followed by adding Glu only) was similar for both mea-sures. Also, the prolonged time with increased bacterial growthrates even after 3 months was not reflected in a continuous increasein bacterial PLFAs. Similar discrepancies were earlier reported afteradding plant substrates to soil, where adding alfalfa meal increasedbacterial growth rates even after 2 months incubation with noconcomitant increase in bacterial PLFAs (Rousk and Bååth, 2007a).The authors discussed possible explanations for this, e.g., bacterialPLFAs including large amounts of inactive biomass, different turn-overs of bacterial and fungal PLFAs, and the extent of predationdiffering between fungi and bacteria. Bacterial predators are known

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 biomasscould well be found, even if bacterial growth and production isincreased.

4.6. Comparison between Leu and TdR incorporation

The Leu:TdR incorporation ratio has been found to be positivelycorrelated to bacterial activity in aquatic ecosystems (Servais,1992). In a recent study, Longnecker et al. (2006) also found thathigher Leu:TdR incorporation ratios were associated with the moreactive members of bacterioplankton, that is, those with high nucleicacid content. Similar results were found in the present study, in thatthe treatments leading to the highest increase in bacterial activitiesexhibited the highest Leu:TdR incorporation ratios (Fig. 3). Vintenet al. (2002) added glucose to a soil and found an eightfold increasein TdR incorporation rates, while Leu incorporation increased 16times compared with the unamended control. Increasing Leu:TdRincorporation ratios were also found after substrate (alfalfa andstraw) additions (Rousk and Bååth, 2007a). Thus, an increasedLeu:TdR incorporation ratio with higher bacterial growth ratesappears 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 containingmore protein, since the protein content of a cell is correlated to thecell volume (Simon and Azam, 1989). This suggests that the Leuincorporation technique would be more sensitive in measuring theeffects of additives that increase bacterial growth than the TdRincorporation technique.

However, during the first week after adding gelatine, the Leu:TdRincorporation ratio was low despite high bacterial growth rates(Figs. 3 and 1E). This could be due to an isotope dilution effectresulting from increased amino acid concentrations in the soilarising from the degradation of gelatine during this period. Althoughearlier results have shown isotope dilution for Leu incorporation tobe rather constant in soil (Bååth,1998), this is apparently not the casewhen large amounts of proteins are added. Thus, when addinga protein-containing substrate to soil, the interpretation of Leu in-corporation rate as growth may result in underestimation of the truegrowth rates. Combining the Leu and TdR incorporation techniquesis a good way of elucidating such confounding effects.

4.7. Nutrient limitation

Bacterial growth was limited by a lack of carbon throughout theincubation period in the control soil (Fig. 5), as was the case in thetreatments where nitrogen was added. However, we expected thatadding only large amounts of carbon (Glu and Cell treatments)would cause nitrogen limitation of the bacteria. This was the case inthe short-term for glucose addition (cellulose addition not in-vestigated), but after 3 months of incubation the addition of neitherglucose-C nor nitrogen affected bacterial growth. One explanationof this may be that both carbon and nitrogen were close to limitingbacterial growth, and adding both simultaneously would benecessary to increase growth. This situation has been observedpreviously in some soils (Demoling et al., 2007). However, thebacteria 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 whenit was nitrogen limited. In the former case growth rates increasedrapidly, but in the latter the increase in microbial biomass uponadding nitrogen was also controlled by the flow of carbon from soilorganic matter. This is most easily explained in the situation withcellulose. Although this may induce nitrogen limitation of bacterial

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–23432342

growth, adding nitrogen would not result in an immediate increasein bacterial growth. Exoenzymes produced by the fungi must firstdegrade the cellulose to products available to the bacteria. It is lessevident for the glucose treatment. The glucose added may, how-ever, have been transformed into other substances that were notimmediately available during the 3-month incubation period, forexample to storage products such as poly-beta-hydroxybutyrate.

5. Conclusions

In conclusion, we have shown that both bacteria and fungi werefavoured by an easily available carbon source, although with a dif-ferent time frame, while fungi were initially more favoured bygrowth on the complex substrate cellulose. Nitrogen addition didnot favour bacterial growth, but allowed the group that was alreadyfavoured by the complexity of the substrate to grow better, i.e.,adding nitrogen together with cellulose favoured fungi, whileadding nitrogen together with glucose favoured bacteria. Fungalgrowth on cellulose facilitated the growth of bacteria, while in-creased growth of bacteria had an antagonistic effect on fungalgrowth. Finally, fungal growth appeared to be better correlated torespiration rates than bacterial growth, indicating that the formergroup was more important for decomposition. The use of themeasures indicating bacterial growth and fungal growth allowedrapid and sensitive estimations of changes in growth, in both in-creasing and decreasing phases. Also, the possibility of combiningthese methods, applying conversion factors to calculate biomassproduction (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 ResearchCouncil to E.B.

References

Alden, L., Demoling, F., Bååth, E., 2001. Rapid method of determining factors limitingbacterial growth in soil. Applied and Environmental Microbiology 67, 1830–1838.

Anderson, J., Domsch, K.H., 1973. Quantification of bacterial and fungal contribu-tions to soil respiration. Archiv fur Mikrobiologie 93, 113–127.

Bååth, E., 1992. Thymidine incorporation into macromolecules of bacteria extractedfrom soil by homogenization-centrifugation. Soil Biology & Biochemistry 24,1157–1165.

Bååth, E., 1994. Measurement of protein synthesis by soil bacterial assemblageswith the leucine incorporation technique. Biology and Fertility of Soils 17,147–153.

Bååth, E., 1998. Growth rates of bacterial communities in soil at varying pH:a comparison of the thymidine and leucine incorporation techniques. MicrobialEcology 36, 316–327.

Bååth, E., 2001. Estimation of fungal growth rates in soil using 14C-acetateincorporation into ergosterol. Soil Biology & Biochemistry 33, 2011–2018.

Bååth, E., Lohm, U., Lundgren, B., Rosswall, T., Soderstrom, B., Sohlenius, B., Wiren, A.,1978. The effect of nitrogen and carbon supply on the development of soilorganism populations and pine seedlings: a microcosm experiment. Oikos 31,153–163.

Bååth, E., Pettersson, M., Soderberg, K.H., 2001. Adaptation of a rapid andeconomical microcentrifugation method to measure thymidine and leucineincorporation by soil bacteria. Soil Biology & Biochemistry 33, 1571–1574.

Bailey, V.L., Smith, J.L., Bolton, H., 2003. Novel antibiotics as inhibitors for theselective respiratory inhibition method of measuring fungal:bacterial ratios insoil. Biology and Fertility of Soils 38, 154–160.

Bardgett, R.D., Lovell, R.D., Hobbs, P.J., Jarvis, S.C., 1999. Seasonal changes in soilmicrobial communities along a fertility gradient of temperate grasslands. SoilBiology & Biochemistry 31, 1021–1030.

Berg, B., Laskowski, R., 2006. Decomposers: soil microorganisms and animals.Advances in Ecological Research 38, 73–100.

Bittman, S., Forge, T.A., Kowalenko, C.G., 2005. Responses of the bacterial and fungalbiomass in a grassland soil to multiyear applications of dairy manure slurry andfertilizer. Soil Biology & Biochemistry 37, 613–623.

Bjørnlund, L., Christensen, S., 2005. How does litter quality and site heterogeneityinteract on decomposer food webs of a semi-natural forest? Soil Biology &Biochemistry 37, 203–213.

Bouwman, L.A., Bloem, J., Romkens, P.F.A.M., Japenga, J., 2005. EDGA amendment ofslightly heavy metal loaded soil affects heavy metal solubility, crop growth andmicrobivorous nematodes but not bacteria and herbivorous nematodes. SoilBiology & Biochemistry 37, 271–278.

Christensen, S., Bjornlund, L., Vestergard, M., 2007. Decomposer biomass in therhizosphere to assess rhizodeposition. Oikos 116, 65–74.

De Boer, W., Klein Gunnewiek, P.J.A., Lafeber, P., Janse, J.D., Spit, B.E., Woldendorp, J.W.,1998. Anti-fungal properties of chitinolytic dune soil bacteria. Soil Biology &Biochemistry 30, 193–203.

De Boer, W., Klein Gunnewiek, P.J.A., Kowalchuk, G.A., van Veen, J., 2003. Microbialcommunity composition affects soil fungistasis. Applied and EnvironmentalMicrobiology 69, 835–844.

De Boer, W., Folman, L.B., Summerbell, R.C., Boddy, L., 2005. Living in a fungal world:impact of fungi on soil bacterial niche development. FEMS MicrobiologyReviews 29, 795–811.

De Boer, W., Wagenaar, A.-M., Klein Gunnewiek, P.J.A., van Veen, J.A., 2007. In vitrosuppression of fungi caused by combinations of apparently non-antagonisticsoil bacteria. FEMS Microbiology Ecology 59, 177–185.

De Ruiter, P.C., van Veen, J.A., Moore, J.C., Brussard, L., Hunt, H.W., 1993. Calculationof nitrogen mineralization in soil food webs. Plant and Soil 157, 263–273.

De Vries, F.T., Hoffland, E., van Eekeren, N., Brussard, L., Bloem, J., 2006. Fungal/bacterial ratios in grasslands with contrasting nitrogen management. SoilBiology & Biochemistry 38, 2092–2103.

Del Giorgio, P.A., Cole, J.J., 1998. Bacterial growth efficiency in natural aquaticsystems. Annual Review of Ecology and Systematics 29, 503–541.

Demoling, F., Figueroa, D., Bååth, E., 2007. Comparisons of factors limiting bacterialgrowth in different soils. Soil Biology & Biochemistry 39, 2485–2495.

Dilly, O., 2004. Effects of glucose, cellulose, and humic acids on soil microbial eco-physiology. Journal of Plant Nutrition and Soil Science 167, 261–266.

Engelking, B., Flessa, H., Joergensen, R.G., 2007. Shifts in amino sugar and ergosterolcontents after addition of sucrose and cellulose to soil. Soil Biology & Bio-chemistry 39, 2111–2118.

Ferris, H., Bongers, T., 2006. Nematode indicators of organic enrichment. Journal ofNematology 38, 3–12.

Fischer, H., Mille-Lindblom, C., Zwirnmann, E., Tranvik, L.J., 2006. Contribution offungi and bacteria to the formation of dissolved organic carbon from decayingcommon reed (Phragmitis australis). Archiv fur Hydrobiologie 166, 79–97.

Frostegård, Å, Bååth, E., 1996. The use of phospholipid fatty acid analysis to estimatebacterial and fungal biomass in soil. Biology and Fertility of Soils 22, 59–65.

Frostegård, Å., Tunlid, A., Bååth, E., 1993. Phospholipid fatty acid composition,biomass and activity of microbial communities from two soil types experi-mentally exposed to different heavy metals. Applied and EnvironmentalMicrobiology 59, 3605–3617.

Gulis, V., Suberkropp, K., 2003. Interactions between stream fungi and bacteriaassociated with decomposing leaf litter at different levels of nutrient avail-ability. Aquatic Microbial Ecology 30, 149–157.

Hu, S., van Bruggen, A.H.C., 1997. Microbial dynamics associated with multiphasicdecomposition of 14C-labeled cellulose in soil. Microbial Ecology 33, 134–143.

Jensen, K.D., Beier, C., Michelsen, A., Emmett, B.A., 2003. Effects of experimentaldrought on microbial processes in two temperate heathlands at contrastingwater conditions. Applied Soil Ecology 24, 165–176.

Longnecker, K., Sherr, B.F., Sherr, E.B., 2006. Variation in cell-specific rates of leucineand thymidine incorporation by marine bacteria with high and low nucleic acidcontent off the Oregon coast. Aquatic Microbial Ecology 43, 113–125.

Mille-Lindblom, C., Tranvik, L.J., 2003. Antagonism between bacteria and fungi ondecomposing aquatic plant litter. Microbial Ecology 45, 173–182.

Mille-Lindblom, C., Fischer, H., Tranvik, L.J., 2006. Antagonism between bacteria andfungi: substrate competition and a possible trade-off between fungal growthand tolerance towards bacteria. Oikos 113, 233–242.

Møller, J., Miller, M., Kjøller, A., 1999. Fungal-bacterial interaction on beech leaves:influence on decomposition and dissolved organic carbon quality. Soil Biology &Biochemistry 31, 367–374.

Moore, J.C., McCann, K., de Ruiter, P.C., 2005. Modelling trophic pathways, nutrientcycling, and dynamic stability in soils. Pedobiologia 49, 499–510.

Newell, S.Y., Fallon, R.D., 1991. Towards a method for measuring instantaneousfungal growth rates in field samples. Ecology 72, 1547–1559.

Olsson, P.A., Chalot, M., Bååth, E., Finlay, R.D., Soderstrom, B., 1996. Ectomycorrhizalmycelia reduce bacterial activity in sandy soil. FEMS Microbiology Ecology 21,77–86.

Paul, E.A., Clark, F.E., 1996. Soil Microbiology and Biochemistry, second ed. AcademicPress, USA.

Pietikainen, J., Pettersson, M., Bååth, E., 2005. Comparison of temperature effects onsoil respiration and bacterial and fungal growth rates. FEMS MicrobiologyEcology 52, 49–58.

Rajapaksha, R.M.C.P., Tobor-Kap1on, M.A., Bååth, E., 2004. Metal toxicity affectsfungal and bacterial activities in soil differently. Applied and EnvironmentalMicrobiology 70, 2966–2973.

Robarts, R.D., Zohary, T., 1993. Fact or fiction – bacterial growth rates and pro-duction as determined by [methyl-3H]thymidine? Advances in MicrobialEcology 13, 371–425.

Romanı, A.M., Fischer, H., Mille-Lindblom, C., Tranvik, L.J., 2006. Interactions ofbacteria and fungi on decomposing litter: differential extracellular enzymeactivities. Ecology 87, 2559–2569.

S. Meidute et al. / Soil Biology & Biochemistry 40 (2008) 2334–2343 2343

Rousk, J., Bååth, E., 2007a. Fungal and bacterial growth in soil with plant materialsof different C/N ratios. FEMS Microbiology Ecology 62, 258–267.

Rousk, J., Bååth, E., 2007b. Fungal biomass production and turnover in soil esti-mated using the acetate-in-ergosterol technique. Soil Biology & Biochemistry39, 2173–2177.

Rousk, J., Alden Demoling, L., Bahr, A., Bååth, E., 2008. Examining the fungal andbacterial niche overlap using selective inhibitors in soil. FEMS MicrobiologyEcology 63, 350–358.

Saetre, P., Stark, J.M., 2005. Microbial dynamics and carbon and nitrogen cyclingfollowing re-wetting of soils beneath two semi-arid plant species. Oecologia142, 247–260.

Schimel, J.P., Weintraub, M.N., 2003. The implications of exoenzyme activity onmicrobial carbon and nitrogen limitation in soil: a theoretical model. SoilBiology & Biochemistry 35, 549–563.

Servais, P., 1992. Bacterial production measured by 3H thymidine and 3H leucineincorporation in various aquatic ecosystems. Archiv fur Hydrobiologie 37,73–81.

Simon, M., Azam, F., 1989. Protein content and protein synthesis rates of planktonicmarine bacteria. Marine Ecology Progress Series 51, 201–213.

Tobor-Kap1on, M.A., Bloem, J., Romkens, P.F.A.M., de Ruiter, P.C., 2005. Functionalstability of microbial communities in contaminated soils. Oikos 111, 119–129.

Van der Wal, A., van Veen, J.A., Pijl, A.S., Summerbell, R.C., de Boer, W., 2006.Constraints on development of fungal biomass and decomposition processesduring restoration of arable sandy soils. Soil Biology & Biochemistry 38, 2890–2902.

Vinten, A.J.A., Whitmore, A.P., Bloem, J., Howard, R., Wright, F., 2002. Factorsaffecting N immobilisation/mineralisation kinetics for cellulose, glucose- andstraw-amended sandy soils. Biology and Fertility of Soils 36, 190–199.