bacterial growth and growth-limiting nutrients following chronic nitrogen additions to a hardwood...

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Bacterial growth and growth-limiting nutrients following chronic nitrogen additions to a hardwood forest soil Pramod N. Kamble a, b, c , Johannes Rousk c , Serita D. Frey d , Erland Bååth c, * a P.G. Department of Environmental Science, PVP College Pravanagar, University of Pune, Pune, India b K.T.H.M. College Nashik, University of Pune, Pune, India c Section of Microbial Ecology, Department of Biology, Ecology Building, Lund University, SE-223 62 Lund, Sweden d Department of Natural Resources & the Environment, University of New Hampshire, Durham, NH 03824, USA article info Article history: Received 17 August 2012 Received in revised form 30 November 2012 Accepted 28 December 2012 Available online 22 January 2013 Keywords: N-deposition Limiting factors Bacterial growth Leucine incorporation N availability abstract Increasing nitrogen (N) deposition due to anthropogenic activities has become a signicant global change threat to N-poor terrestrial ecosystems. We compared bacterial growth and nutrients limiting bacterial growth in one of the longest running experiments on increasing N-deposition to a temperate forest, the Chronic Nitrogen Amendment Study at Harvard Forest, USA. Soil samples were collected in fall 2009 from the organic and mineral horizons of plots treated annually since 1988 with 0 (unfertilized), 50 (low N) or 150 (high N) kg N ha 1 as NH 4 NO 3 . In the organic horizon, bacterial growth (leucine incorporation) decreased by 5 times in the high N plots compared to the unfertilized treatment, while no decrease was observed in the mineral horizon. Bacterial growth in all soils was primarily limited by lack of carbon (C), although adding only C (as glucose) resulted in only a minor increase in bacterial growth in the unfer- tilized soil compared to adding C in combination with N. The bacterial growth induced by adding only C increased with higher level of N fertilization, up to 7e8 times the level without any C addition in the high N treatment, suggesting increased availability of N for the bacteria with increasing N addition. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Anthropogenic activities have altered the global and regional cycles of nitrogen (N), increasing N emissions to the atmosphere due to industrialization, agricultural practices, and the combustion of fossil fuels (Vitousek et al., 1997; Aber et al., 2003; Galloway et al., 2008). Atmospheric N emissions are estimated to have increased N deposition in parts of Europe and the US between 5- and 50-fold higher than pre-industrial levels (Dentener et al., 2006), doubling the rate of N input into the terrestrial N cycle, with anthropogenic sources of N currently exceeding natural inputs (Vitousek et al., 1997; Galloway et al., 2008). Due to the historically low availabil- ity of N in boreal and northern temperate forest soils, these systems are particularly sensitive to altered N inputs via N deposition (Kellner and Mårshagen, 1991; Strengbom et al., 2001). The effect of increasing N deposition to soil has usually been experimentally studied by adding N as fertilizer. In forest soils this usually results in decreasing microbial biomass and respiration (Nohrstedt et al., 1989; Frey et al., 2004; Demoling et al., 2008; Pregitzer et al., 2008; Treseder, 2008; Janssens et al., 2010; Liu and Greaver, 2010). One factor that has received little attention is the extent to which N additions to soils normally low in N might shift the limiting resource for microbial growth. Carbon (C) is commonly thought to be the most common limiting nutrient for microbial growth in soil (Anderson and Domsch, 1989; Joergensen and Scheu, 1999; Aldén et al., 2001; Ilstedt and Singh, 2005; Demoling et al., 2007), although N limiting conditions have been reported (Rinnan et al., 2007; Sistla et al., 2012). In boreal forests with high soil C/N ratios, the microorganisms may be close to becoming co- limited by both C and N, or even N limited. Increasing N deposi- tion might therefore result in altered limiting resources for microbial growth in soil. We studied a hardwood stand located at the Harvard Forest Chronic N Amendment Study, where a mixed hardwood stand has been fertilized with 0, 50, or 150 kg N ha 1 for the past 22 years. We expected bacterial growth to be close to being co-limited by both C and N in the unfertilized soil, with more clear C limitation with increasing N amendment levels. We consequently hypothesized that the increased growth of bacteria after adding excess C would * Corresponding author. Tel.: þ46 46 222 42 64. E-mail address: [email protected] (E. Bååth). Contents lists available at SciVerse ScienceDirect Soil Biology & Biochemistry journal homepage: www.elsevier.com/locate/soilbio 0038-0717/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.soilbio.2012.12.017 Soil Biology & Biochemistry 59 (2013) 32e37

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Soil Biology & Biochemistry 59 (2013) 32e37

Contents lists available

Soil Biology & Biochemistry

journal homepage: www.elsevier .com/locate/soi lbio

Bacterial growth and growth-limiting nutrients following chronicnitrogen additions to a hardwood forest soil

Pramod N. Kamble a,b,c, Johannes Rousk c, Serita D. Frey d, Erland Bååth c,*

a P.G. Department of Environmental Science, PVP College Pravanagar, University of Pune, Pune, IndiabK.T.H.M. College Nashik, University of Pune, Pune, Indiac Section of Microbial Ecology, Department of Biology, Ecology Building, Lund University, SE-223 62 Lund, SwedendDepartment of Natural Resources & the Environment, University of New Hampshire, Durham, NH 03824, USA

a r t i c l e i n f o

Article history:Received 17 August 2012Received in revised form30 November 2012Accepted 28 December 2012Available online 22 January 2013

Keywords:N-depositionLimiting factorsBacterial growthLeucine incorporationN availability

* Corresponding author. Tel.: þ46 46 222 42 64.E-mail address: [email protected] (E. Bååth).

0038-0717/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.soilbio.2012.12.017

a b s t r a c t

Increasing nitrogen (N) deposition due to anthropogenic activities has become a significant global changethreat to N-poor terrestrial ecosystems. We compared bacterial growth and nutrients limiting bacterialgrowth in one of the longest running experiments on increasing N-deposition to a temperate forest, theChronic Nitrogen Amendment Study at Harvard Forest, USA. Soil samples were collected in fall 2009 fromthe organic and mineral horizons of plots treated annually since 1988 with 0 (unfertilized), 50 (low N) or150 (high N) kg N ha�1 as NH4NO3. In the organic horizon, bacterial growth (leucine incorporation)decreased by 5 times in the high N plots compared to the unfertilized treatment, while no decrease wasobserved in the mineral horizon. Bacterial growth in all soils was primarily limited by lack of carbon (C),although adding only C (as glucose) resulted in only a minor increase in bacterial growth in the unfer-tilized soil compared to adding C in combination with N. The bacterial growth induced by adding only Cincreased with higher level of N fertilization, up to 7e8 times the level without any C addition in the highN treatment, suggesting increased availability of N for the bacteria with increasing N addition.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Anthropogenic activities have altered the global and regionalcycles of nitrogen (N), increasing N emissions to the atmospheredue to industrialization, agricultural practices, and the combustionof fossil fuels (Vitousek et al., 1997; Aber et al., 2003; Galloway et al.,2008). Atmospheric N emissions are estimated to have increased Ndeposition in parts of Europe and the US between 5- and 50-foldhigher than pre-industrial levels (Dentener et al., 2006), doublingthe rate of N input into the terrestrial N cycle, with anthropogenicsources of N currently exceeding natural inputs (Vitousek et al.,1997; Galloway et al., 2008). Due to the historically low availabil-ity of N in boreal and northern temperate forest soils, these systemsare particularly sensitive to altered N inputs via N deposition(Kellner and Mårshagen, 1991; Strengbom et al., 2001).

The effect of increasing N deposition to soil has usually beenexperimentally studied by adding N as fertilizer. In forest soils this

All rights reserved.

usually results in decreasing microbial biomass and respiration(Nohrstedt et al., 1989; Frey et al., 2004; Demoling et al., 2008;Pregitzer et al., 2008; Treseder, 2008; Janssens et al., 2010; Liu andGreaver, 2010). One factor that has received little attention is theextent to which N additions to soils normally low in N might shiftthe limiting resource for microbial growth. Carbon (C) is commonlythought to be the most common limiting nutrient for microbialgrowth in soil (Anderson and Domsch,1989; Joergensen and Scheu,1999; Aldén et al., 2001; Ilstedt and Singh, 2005; Demoling et al.,2007), although N limiting conditions have been reported(Rinnan et al., 2007; Sistla et al., 2012). In boreal forests with highsoil C/N ratios, the microorganisms may be close to becoming co-limited by both C and N, or even N limited. Increasing N deposi-tion might therefore result in altered limiting resources formicrobial growth in soil.

We studied a hardwood stand located at the Harvard ForestChronic N Amendment Study, where a mixed hardwood stand hasbeen fertilized with 0, 50, or 150 kg N ha�1 for the past 22 years. Weexpected bacterial growth to be close to being co-limited by both Cand N in the unfertilized soil, with more clear C limitation withincreasing N amendment levels. We consequently hypothesizedthat the increased growth of bacteria after adding excess C would

P.N. Kamble et al. / Soil Biology & Biochemistry 59 (2013) 32e37 33

be proportional to available soil N. Finally, we wanted to comparethe effect of the N additions, which are made to the soil surface, onbacterial growth and extent of nutrient limitation in differenthorizons.

2. Materials and methods

2.1. Soils and chemical analyses

Soil sampleswere collected in September 2009 from the ChronicNitrogen Amendment Study at the Harvard Forest Long TermEcological Research (LTER) site in central Massachusetts, USA.These plots were established in 1988 to study long-term effects of Naddition on the forest ecosystem (Aber and Magill, 2004). Soils areof the Gloucester series (fine loamy, mixed, mesic, Typic Dystro-chrepts, USDA). Mean annual temperature is 7 �C and average totalannual precipitation is 1100 mm. We collected samples froma hardwood stand dominated by black (Quercus velutina) and red(Quercus rubra) oakmixedwith black birch (Betula lenta), redmaple(Acer rubrum) and American beech (Fagus grandifolia). There arethree 30 � 30 m plots treated annually with 0 (unfertilized), 50(Low N) or 150 (High N) kg N ha�1 as NH4NO3. Soil samples werecollected from each of four replicate 5 � 5 m sub-plots within eachtreatment plot. Two 8 cm diameter and 10 cm long cores wereremoved from each sub-plot, separated into the organic and min-eral horizon, and bulked by soil horizon. Samples were sieved(2 mm) and stored field moist at 4 �C until analyzed. Soil pH wasmeasured in water extracts (1:1 water:soil). Moisture contentswere determined gravimetrically (24 h at 105 �C), and soil organicmatter (OM) concentrations were estimated as loss on ignition afterheating to 600 �C overnight in a muffle furnace.

2.2. Bacterial growth

Bacterial growth was estimated using the leucine (Leu) incor-poration technique (Kirchman et al., 1985; Bååth, 1994; Bååth et al.,2001), which measures the rate of protein synthesis as a proxy forbacterial growth (Kirchman, 2001). Briefly 1 g of soil was mixedwith 20 mL water and vortexed on a multivortex shaker at max-imum intensity for 3 min. Then the soil samples were subjected tolow speed centrifugation at 1000 g for 10 min creating a bacterialsuspension that was transferred to 2-mL micro-centrifugationtubes. A radioactive precursor, 2 ml 3H Leu (L-4,5-3H-Leucine,37 MBq ml�1, 1.48e2.22 TBq mmol�1, Perkin Elmer, USA) wascombined with non-radioactive Leu and added to each tube, cre-ating a final leucine concentration of 275 nM. After a 2-h incuba-tion, growth was terminated using 75 mL 100% tri-chloroacetic acid.Washing and subsequent measurement of radioactivity were per-formed as described by Bååth et al. (2001). The amount of Leuincorporated into extracted bacteria (pmol h�1 g�1 OM in extractedbacteria) was used as a measure of bacterial growth.

2.3. Nutrient limitation for bacterial growth

Nutrient limitation for bacterial growthwas studied according toAldén et al. (2001) with modifications (Demoling et al., 2007). Fromeach soil sample 1 g of soil was placed in eight 50-ml centrifugetubes with lids. Glucose (C), NH4NO3 (N) and K2HPO4 (P) were thenadded in a full factorial design: No nutrient addition control (Co), C,N, P, CN, CP, NP and CNP. In soils from the organic horizon, 5 mgglucose (equivalent to 2 mg C), 0.142 mg NH4NO3 (equivalent to0.05 mg NH4NO3eN) and 0.112 mg K2HPO4 (equivalent to 0.02 mgK2HPO4eP) were added per gram soil. In soils from the mineralhorizon, 2.5 mg glucose, 0.142 mg NH4NO3 and 0.112 mg K2HPO4were used per gram soil. These amounts had been chosen from an

earlier test,whereadditions of up to40mgglucose, 4.56mgNH4NO3and 3.6 mg K2HPO4 per gram soil were tested. However, highamounts of nutrients inhibited bacterial growth, especially whenaddingNH4NO3 to soil from the organic horizon. The soilswere thenincubated for 72h at roomtemperature (approx. 22 �C) andbacterialgrowth was estimated with the leucine incorporation technique(see above). The selected addition rates of nutrients resulted ina high growth in the CNP treatment, without resulting in any sig-nificant decrease in other nutrient addition combinations.

Alleviating a nutrient limitation will increase the bacterialgrowth rate resulting in an increased bacterial number. Thus, theeffect of adding a limiting nutrient can both be an increased growthrate and an increased biomass. The leucine incorporation methodas performed here does not differentiate between these twomechanisms, but both will result in increased leucine incorpo-ration, with the conclusion that the increase was due to thenutrient addition.

2.4. Statistics

Comparison of bacterial growth in the different long-term Naddition treatments was made by one-way ANOVA for each hori-zon, followed by Tukey’s test (p < 0.05). For the data on limitingfactors, each treatment and horizon was initially subjected to a 3-factor ANOVA (factors C, N and P addition with interactions) witheach replicate soil sample as block. The data were logarithmicallytransformed to stabilize the variance. In no case was the addition ofP significant. Therefore, only C and N treatments were comparedwith the control using Dunnett’s post hoc test. Means and SE wereplotted after antilog transformation of the data (Fig. 1).

To indicate the extent of C limitation (and thus the availability ofN to soil bacteria, i.e. when the primary C-limitationwas relieved bysurplus addition of glucose-C), the effect of adding C (the C/Cobacterial growth ratio, i.e. bacterial growth with added C/bacterialgrowth in the control without added C) was calculated and com-pared to the effect of adding only N (the N/Co ratio, i.e. the bacterialgrowth with added N/bacterial growth without added N). The extragrowth induced by adding Nwhen the C limitationwas removed byadding excess C as glucose was expressed by calculating the CN/Cgrowth ratio (i.e. bacterial growth with added CN/bacterial growthwith only added C).

3. Results

Bacterial growth in the unamended soils was higher in soilsfrom the organic than from the mineral horizon when expressedboth per g of soil (data not shown) and per g OM (Table 1). The highN treatment significantly decreased bacterial growth five fold in theorganic horizon compared to the unfertilized treatment, while inthe mineral horizon there was significantly higher bacterial growthin the low N compared to the other two treatments (p < 0.05).

The effect on bacterial growth of adding different nutrients tosoil in short-term assays was similar in the mineral and organichorizons (Fig. 1). There was no effect on bacterial growth of addingonly P or P in combination with N (data not shown). Mean relativeeffects, normalized to 1 for the no addition control, over all fertilizertreatments and both organic andmineral horizons, were 1.15� 0.10and 1.07 � 0.03 for P and NP additions, respectively. Adding P incombination with C also did not result in any difference comparedto similar additions without P (comparing C vs CP and CN vs CNP).Thus, since adding P did not affect bacterial growth, mean values ofC and N effects were calculated over both P treatments.

Adding both C and N (the CN treatment) always resulted inincreased growth of the bacteria compared to the no additioncontrol, increasing around 2.5e5 times in the organic horizon

Table 1Bacterial growth, pH and organic matter (OM) in soils from organic and mineralhorizons with chronic addition of N as NH4NO3. Unfert. ¼ unfertilized with no Naddition, Low N ¼ addition of 50 kg N ha�2 since 1988, High N ¼ addition of150 kg N ha�1 since 1988. � SE, n ¼ 4. F-values from ANOVAs are shown, and thesame letter indicates no significant differences (Tukey’s, p < 0.05) between treat-ments within the same horizon. # ¼ ANOVA was made on log-transformed values.

Horizon Treatment pH OM (g/g) Bacterialgrowth (pmolLeu h�1 g�1 OM)#

Org. Unfert. 5.1 � 0.23a 0.47 � 0.08a 203 � 57a

Low N 5.0 � 0.05a 0.45 � 0.03a 213 � 83a

High N 4.4 � 0.03b 0.38 � 0.10a 38 � 13b

Min. Unfert. 5.0 � 0.08a 0.152 � 0.009a 85 � 7.3a

Low N 5.2 � 0.08a 0.142 � 0.011a 129 � 7.1b

High N 4.4 � 0.08b 0.135 � 0.012a 74 � 10.0a

F Org. 4.5 (p ¼ 0.049) n.s. (p ¼ 0.69) 5.9 (p ¼ 0.027)Min. 30.0 (p < 0.001) n.s. (p ¼ 0.55) 12.7 (p ¼ 0.0024)

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Fig. 1. Bacterial growth, estimated using leucine incorporation, 72 h after adding C(as glucose) and N (as NH4NO3) in a full factorial design (Control indicates no nutrientaddition) in soils from the (A) organic and (B) mineral horizons from a hardwood forestwith chronic N-fertilization as NH4NO3. Bacterial growth was normalized(Control ¼ 1). Unfertilized ¼ soil with no fertilizer addition, Low N ¼ addition of50 kg N ha�1 since 1988, High N ¼ addition of 150 kg N ha�1 since 1988. Bars indicateSE.

P.N. Kamble et al. / Soil Biology & Biochemistry 59 (2013) 32e3734

(Fig. 1A) and around 10e15 times in the mineral horizon (Fig. 1B).Thus, the amount of C and N added was enough to induce anincreased bacterial growth in all soils.

Adding only N did not result in increased growth in any of thefertilizer treatments, with a mean value of 0.97 � 0.04 compared tothe no addition control. In the unfertilized soil only the CN treat-ment had a significant higher bacterial growth compared to the noaddition control (p < 0.001). However, the C treatment increasedbacterial growth by 25 and 40% compared to the no addition con-trol in the organic and mineral horizon, respectively, while Naddition decreased bacterial growth slightly. This suggests thatbacterial growth was limited by lack of C in the unfertilized soil. Inthe soil from the Low N organic horizon there was significantlyincreased bacterial growth compared to the no addition controlboth in the C and CN treatment (p < 0.05 and 0.001, respectively),which also was the case in the mineral horizon (p < 0.001 for bothhorizons). In both cases adding only C increased bacterial growth2.5e3 times that of the no addition control, while CN additionincreased it around 4 and 10 times in the organic and mineralsoil, respectively. In the High N fertilization adding only Cincreased growth to around 7e8 times the level in the noaddition control in both soil horizons (p < 0.001 for bothhorizons). CN addition resulted in higher bacterial growth thanonly adding C in the soils from the mineral horizon. In the soilsfrom the organic horizon adding CN resulted in lower growthcompared to only C in the High N soils.

To simplify the comparison of C and N limitation we alsoplotted the log C/Co and the log N/Co ratios (bacterial growth withaddition of C or N, respectively, divided with bacterial growth inthe no addition control) in the different fertilizer treatments(Fig. 2A and B). The log N/Co ratio was not significantly differentfrom unity in any of the fertilizer treatments (Fig. 2B), clearlyshowing that lack of N was not limiting bacterial growth. The logC/Co ratio, on the other hand, was higher than 1 in all soils sug-gesting a consistent C limitation. The log C/Co ratio also increasedwith fertilizer addition both in the mineral and organic horizon(Fig. 2A). That is, the level of bacterial growth-limitation by lack ofC increased with the level of long-term N-fertilization. The effectof increasing long-term N-fertilization could also be seen by thedecreasing level of extra growth of bacteria induced by combiningC with N, i.e. a decreasing need for the added N with increasinglevel of N-fertilization in soils from both depths (the log CN/Cratio; Fig. 2C). However, in this case the organic and mineral ho-rizon differed in their response; the bacterial growth being lessresponsive to the CN treatment in soils from the organic horizonthan in the mineral horizon. In the high N treatment there waseven a negative effect of adding N together with C compared toonly C.

4. Discussion

The high level of chronic fertilization (High N) decreased bac-terial growth drastically in the organic horizon (Table 1). Demolinget al. (2008) also found that bacterial growth, estimated usingthymidine incorporation, was negatively affected in N-fertilizedconiferous forest humus. Thus, there seems to be a recurringpattern with decreased bacterial growth after N-fertilization intemperate forest soils, in parallel with the well-known decrease inmicrobial biomass and respiration (see Introduction), indicatinga decrease in resource availability. However, this negative effect onbacterial growth was more evident in the organic horizon than inthe mineral horizon, where no significant effect was found. This isin accordance with a cascading effect as fertilizer N has movedthrough the soil after the surface application. McDowell et al.(2004) found a high concentration of N in lysimeter water from

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Fig. 2. Bacterial growth, estimated using leucine incorporation, 72 h after adding C (asglucose) and N (as NH4NO3) to soils from organic (Org.) and mineral (Min.) horizonsfrom a hardwood forest with chronic N-fertilization as NH4NO3. Co indicates the nonutrient addition control. Comparison of (A) the level of growth stimulation by addingC compared to the no nutrient addition control (log C/Co), (B) the level of growthstimulation by adding N compared to the no nutrient addition control (log N/Co), and(C) extra bacterial growth by combining C and N compared to only adding C (log CN/C).Unfertilized ¼ soil with no fertilizer addition, Low N ¼ addition of 50 kg N ha�1 since1988, High N ¼ addition of 150 kg N ha�1 since 1988. Bars indicate SE. Thin line in-dicates no effects.

P.N. Kamble et al. / Soil Biology & Biochemistry 59 (2013) 32e37 35

the High N plots already in 1993, while similar levels in themineral horizon were not consistently found until after 1998(Magill et al., 2004). Thus, the organic horizon was affected soonerafter initiation of the N-fertilization than the mineral horizon,possibly resulting in a more drastic effect on the soilmicroorganisms.

Soil pH decreased in the High N treatment of the organic ho-rizon (Table 1), and it is well-known that low pH in soil decreasesthe competitive ability of bacteria compared to fungi (Bååth, 1998;Rousk et al., 2009, 2011; Fernández-Calviño et al., 2011). Areduction of soil pH was also suggested as a possible mechanismto explain that bacterial growth was more negatively affectedthan fungal growth by Demoling et al. (2008) in N-fertilized for-ests. However, this acidification is unlikely to be the soleexplanation for the reduction of bacterial growth in the High N-fertilized soils. Soil pH decreased to the same extent in the min-eral and the organic soil horizon, while the decrease in bacterialgrowth was only found in the organic horizon (Table 1). Fur-thermore, in one of the soils studied by Demoling et al. (2008)there was no evidence for N-fertilization inducing a decreasedpH, although bacterial growth was halved compared to the un-fertilized treatment.

Bacterial growth in the unfertilized soil was limited by lack ofC, while adding N or P had no effect (Fig. 1). This is in accordancewith most soils reported to have C as the primary limiting sub-stance for microbial growth (Nordgren, 1992; Joergensen andScheu, 1999; Aldén et al., 2001; Ilstedt and Singh, 2005;Demoling et al., 2007; Göransson et al., 2011). This includestemperate forest soils (Ekblad and Nordgren, 2002; Demolinget al., 2008), although they are considered to be low in N, withtree growth mainly limited by lack of N (Attiwill and Adams, 1993;LeBauer and Treseder, 2008). However, adding C without N in thepresent study resulted in only a minor increase in bacterialgrowth in the unfertilized soil, consistent with the resultsobtained by Demoling et al. (2008). Demoling et al. (2008)reported an increase in the bacterial growth of between 20 and40% in three Swedish coniferous forests, compared with thepresent study where we report 25e40% increases depending onsoil horizon (Fig. 1). Thus, the unfertilized soils in these studiesappeared to be in a situation, where relieving the limitation of theprimary limiting factor for growth (C) through surplus additionrapidly induced limitation by the secondary limiting factor (N)with the net result of only a minor increase in bacterial growth.This situation is close to what has been referred to as “co-limi-tation” and has recently been reported from similar bioassays inboth soil (Yoshitake et al., 2007; Göransson et al., 2011) and waterecosystems (Elser et al., 2009a, 2009b).

In all soils, except in the organic horizon from the high Ntreatment, adding N together with C in the short-term assayincreased bacterial growthmore than adding only C (Figs.1 and 2C),while adding P had no effect, even at higher concentrations. Thus, Nwas the secondary limiting substance in these soils, which wasexpected for these N-poor soils. Only at the highest level of Naddition in the soil from the organic horizon was there an indica-tion of N not being the secondary limiting substance. However, theaddition of N can be problematic in that especially in organic forestsoil horizons, high levels of N can immediately result in inhibitedbacterial growth (Aldén et al., 2001). This was also indicated by ourpreliminary studies where adding higher concentrations of Nresulted in decreased bacterial growth in all soils (data not shown).It is thus possible that in the high N treatment in the organic ho-rizon, the addition of extra N together with already high amounts ofavailable N in the soil resulted in decreased bacterial growth duringthe 72 h incubation. This was most obvious after adding CN(Fig. 2B), but was also suggested by a tendency for decreased

P.N. Kamble et al. / Soil Biology & Biochemistry 59 (2013) 32e3736

bacterial growth, although not significantly so, when adding only N(Figs. 1A and 2B, High N treatment).

Whenadding theprimary limiting nutrient in excess, growthwillincreaseuntil it is limitedby the secondary limitingnutrient (Liebig’sLaw of theMinimum). The extent of growth on the primary limitingnutrient will thus indicate the availability of the second limitingnutrient; extensive growth will only be possible in the presence ofa large amount of the secondary limiting nutrient. In our case theprimary nutrientwas C and the secondary nutrientwas N in all soils.The level of the bacterial response to adding C (the C/Co ratio)increased with the fertilization level, being low in the unfertilizedsoil and high in the high N treatment (Fig. 2A). This suggests anincreasing bacterial availability to N in soils with higher N fertiliza-tion rates. We did not measure the content of available N in thesesoils, but a close correlation between the bacterial growth responseand previously published lysimeter concentrations of dissolved N inthe soil samples (10 year means of dissolved N in water from zerotension lysimeters installed beneath the forest floor; Table 2 inMcDowell et al. (2004)) is suggestive. The variation in bacterialgrowth response after adding C was also used by Göransson et al.(2011) to argue that N availability increased with soil age alonga chronosequence from a glacier forefield, and by Demoling et al.(2008) to suggest increasingN availability in N-fertilized forest soils.

Increasing N loads resulted in reduced bacterial growth, indicat-ing a reduction in the availability of growth-limiting resources. Thisdecrease was, however, not due to a switch in the limiting nutrientsfor growth. Instead the present results suggest that bacterial growthwas primarily limited by C in all the soils. Chronic, long-term N-fer-tilization only resulted in changing soil from having a bacterialgrowth initially beingC-limited, but close to being co-limitedbybothC and N, into soils with a more ample supply of available N for bac-terial growth. N fertilization thus had a two-pronged effect on bac-terial growth: (i) adirecteffectonNavailability,whichwas increased,and (ii) an indirect effect on C availability, which decreased. Botheffects increased the strength of limitation by C, while only the latterresulted in an overall reduction in bacterial growth.

Acknowledgments

This studywas supportedbyanErasmusMundi grant toP.N.K., bythe Swedish Research Council to E.B. (Project No. 621-2009-4503)and to J.R. (Project No. 621-2011-5719), by the U.S. National ScienceFoundation to S.D.F (Project No. 0620443) and by the U.S. NationalScience Foundation Long-term Ecological Research (LTER) Programthatprovided support for theChronicNitrogenAmendment StudyatHarvard Forest. Thisworkwas part of LUCCI (LundUniversity Centrefor Studies of Carbon Cycle and Climate Interactions).

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