Consistent effects of nitrogen amendments on soil microbial communities and processes across biomes

Consistent effects of nitrogen amendments on soil microbial communities and processes across biomesK E L L Y S.R A M I R E Z*,J O S E P H M.C R A I N E†and NOAH FIERER*‡*Department of Ecology and Evolutionary Biology,University of Colorado,Boulder,80309,USA,†Division of Biology,Kansas State University,Manhattan,Kansas66506,USA,‡Cooperative Institute for Research in Environmental Sciences,University of Colorado,Boulder,Colorado80309,USAAbstractEcosystems worldwide are receiving increasing amounts of reactive nitrogen(N)via anthropogenic activities with the added N having potentially important impacts on microbially mediated belowground carbon dynamics. However,a comprehensive understanding of how elevated N availability affects soil microbial processes and community dynamics remains incomplete.The mechanisms responsible for the observed responses are poorly resolved and we do not know if soil microbial communities respond in a similar manner across ecosystems.We col-lected28soils from a broad range of ecosystems in North America,amended soils with inorganic N,and incubated the soils under controlled conditions for1year.Consistent across nearly all soils,N addition decreased microbial respiration rates,with an average decrease of11%over the year-long incubation,and decreased microbial biomass by 35%.High-throughput pyrosequencing showed that N addition consistently altered bacterial community composi-tion,increasing the relative abundance of Actinobacteria and Firmicutes,and decreasing the relative abundance of Acidobacteria and Verrucomicrobia.Further,N-amended soils consistently had lower activities in a broad suite of extracellular enzymes and had decreased temperature sensitivity,suggesting a shift to the preferential decomposition of more labile C pools.The observed trends held across strong gradients in climate and soil characteristics,indicating that the soil microbial responses to N addition are likely controlled by similar wide-spread mechanisms.Our results support the hypothesis that N addition depresses soil microbial activity by shifting the metabolic capabilities of soil bacterial communities,yielding communities that are less capable of decomposing more recalcitrant soil carbon pools and leading to a potential increase in soil carbon sequestration rates.Keywords:anthropogenic change,bacteria,extracellular enzymes,microbial activity,pyrosequencing,soil decomposition, temperature sensitivityReceived11November2011and accepted19December2011IntroductionNitrogen(N)inputs from anthropogenic sources are currently estimated to be30%to50%greater than those from natural terrestrial sources and tenfold greater than anthropogenic inputs from100years ago(Galloway et al.,2008;Schlesinger,2009;Canfield et al.,2010). Understanding how this additional N will impact ter-restrial ecosystems is becoming increasingly important within the context of the terrestrial carbon(C)budget (Singh et al.,2010;Zaehle et al.,2010).Since N is a limit-ing nutrient to plants in most terrestrial ecosystems (Vitousek et al.,2002),anthropogenic N enrichment can often have strong effects on aboveground primary productivity and plant community composition (Tilman,1987;Gough et al.,2000;Vitousek et al.,2002). In contrast,the belowground microbial responses to elevated N inputs are less well understood.Recent work indicates that N additions can alter the diversity and composition of soil microbial communities(Allison et al.,2008;Campbell et al.,2010;Ramirez et al.,2010b; Bates et al.,2011)though it remains unclear if microbial community shifts are predictable and consistent across ecosystems as few experimental studies have examined responses across a wide range of soil or ecosystem types.Additionally,withfield-based studies it is diffi-cult to determine if microbial community shifts are a direct result of the N addition or an indirect result of changes to plant communities and their C inputs to soils(Waldrop et al.,2004;Ramirez et al.,2010b). Nitrogen enrichments not only influence microbial community composition but also can strongly impact microbial C dynamics–though not in ways that would be predicted from traditional limitation theory (Vitousek&Howarth,1991)as N enrichments typically decrease microbial activity.Strong reductions in soil microbial activities have been documented in bothfield and lab-based studies with the magnitude of the reduc-tion in soil respiration and microbial biomass stronglyCorrespondence:Kelly S.Ramirez,tel.+3034922099,fax+3034921149,e-mail:kelly.ramirez@1918©2012Blackwell Publishing Ltd Global Change Biology(2012)18,1918–1927,doi:10.1111/j.1365-2486.2012.02639.xrelated to the duration and amount of N added to an ecosystem(Treseder,2008;Janssens et al.,2010),with reductions in soil respiration regardless of the form of N added(Ramirez et al.,2010a).Likewise,microbial enzyme activities can be highly sensitive to increases in N,especially those enzymes that degrade complex C compounds(Sinsabaugh et al.,2005;Waldrop&Zak, 2006).Further,Craine et al.(2007)found that N addi-tions decrease recalcitrant-C mineralization,causing an overall decline in soil decomposition rates.This com-monly observed suppression of microbial activity under N enrichment could be considered somewhat counterintuitive given that plant productivity generally increases with addition of N(Gough et al.,2000),which would likely increase C inputs to soils.As traditional nutrient limitation theory does not explain declines in soil microbial activity,a number of hypotheses have been proposed to explain this para-dox.For example,N additions may have indirect effects on microbial communities by altering plant community composition and/or the amounts and types of plant C inputs to soils,reducing microbial activity levels(Meier &Bowman,2008).Other hypotheses involve N-induced shifts in microbial physiologies and/or microbial com-munity compositions that are directly associated with increased soil N availability.For example,the enzyme inhibition hypothesis proposes that N additions directly inhibit those enzymes needed for the decomposition of recalcitrant C thereby reducing overall microbial activ-ity(Fog,1988;Gallo et al.,2004).Similarly,the N-mining hypothesis suggests that under N enrichment,microbes reduce decomposition of recalcitrant C in response to their lowered N requirements(microbes no longer ‘mine’recalcitrant organic matter to obtain N),result-ing in a shift towards labile C decomposition and reduced overall microbial activity(Moorhead&Sinsab-augh,2006;Craine et al.,2007).This shift might occur at the level of the individual microbe and/or be a conse-quence of shifts in the relative abundances of specific microbial taxa.A similar hypothesis,the copiotrophic hypothesis,suggests that N additions decrease micro-bial activities by directly shifting microbial community composition(Fontaine et al.,2003;Fierer et al.,2007, 2011;Ramirez et al.,2010a,b).Under this hypothesis, increases in N reduce the relative abundance of oligo-trophic taxa(those taxa that are adept at catabolizing more recalcitrant C pools)as the alleviation of N limita-tion allows them to be out-competed by more copio-trophic taxa(those taxa that have higher N demands and are more restricted in their catabolic capabilities, specializing on more labile C pools).To identify if N enrichment elicits consistent responses in microbial activity and community compo-sition across ecosystems and to determine what mechanisms may be responsible for the observed responses,soils were collected from28sites throughout North America representing a broad range of biomes and soil types.Soils were amended with inorganic N and incubated for1-year.By incubating the soils under standardized,laboratory conditions we eliminated the indirect effects of N additions on plant C inputs and instead focused only on direct N responses across the We examined effects of N amendments on the soil bacterial community composition,but did not examine effects on the fungal communities,even though such effects may be important,particularly with regard to the process responses examined here(Allison et al., 2008;Edwards et al.,2011;Fontaine,2011).We tested the validity of the various hypotheses outlined above by assessing N effects on soil respiration,microbial bio-mass,bacterial community composition,extracellular enzyme activities and soil temperature sensitivity after the1-year incubation under controlled conditions. MethodsSite and soil descriptionSoils were collected from28sites across North America,rang-ing from17.97to68.63°N and representing a range of cli-mates(mean annual temperatures fromÀ10.3to25.7°C and mean annual precipitation from17.3to300cm)(Fig.1, Table1).The collected soils also represented a broad range of edaphic characteristics(e.g.pH from4.24to8.32,soil C:N from7.62to36.94,and%silt+clay from5to62.5)(Table1). At each site,soil was collected from the top10cm of the A horizon,sieved to2mm and stored at5°C for10–60days until incubations began.Soil C and N concentrations,pH and texture were measured using methods described in Craine et al.(2010).Soil incubation,respiration and microbial biomassTo determine the response of soil microbial communities to N addition,subsamples of soils(20g dry-equivalent)were weighed out into50ml polyethylene centrifuge tubes(Craine et al.,2010).Subsamples were kept at ambient N levels(no N added)or treated with5mg N in the form of NH4NO3,which is approximately equivalent to adding125g N mÀ2to a50-cm soil profile,an amount similar to that applied in manyfield studies(Treseder,2008;Ramirez et al.,2010b).All samples were adjusted to35%water-holding capacity and incubated at 20°C in a humid environment with soil moisture held con-stant through the incubation.Soil respiration was measured 15times over the365-day incubation period(Craine et al., 2010).Briefly,at each measurement,the two replicates of each soil sample were sealed with a gas-tight cap equipped with a rubber septum and incubated for18–72h.At the end of this period,CO2concentrations were measured using a Licor6252 infrared gas analyzer(LiCor,Lincoln,NE,USA)with respiration©2012Blackwell Publishing Ltd,Global Change Biology,18,1918–1927CONSISTENT NITROGEN EFFECTS ON SOILS ACROSS ECOSYSTEMS1919rates determined from the net accumulation of CO2over time. Respiration from ambient samples was used as an estimation of available C,where we assumed that in the absence of mois-ture limitation and at a constant temperature,the rate of CO2 production corresponds to the amount of C readily available for microbial mineralization(Fierer et al.,2003).After the365-day incubation,microbial biomass was mea-sured using the substrate induced respiration(SIR)approach (Wardle&Ghani,1995;Fierer et al.,2003).Briefly,replicates were placed at20°C for48h,at which point400mg of glu-cose was added to one replicate of each soil,the tubes capped after20min of equilibration,and respiration rates(as described above)were determined after2–4h.Bacterial community compositionTo characterize bacterial community composition from each of the28soil samples at the end of the365-day incubation soils were harvested,frozen and stored atÀ20°C for1month.We then extracted DNA using the MoBio PowerSoil DNA extrac-tion kit following the manufacturer’s instructions with modifi-cations described previously(Lauber et al.2009).A portion of the16S rRNA gene was amplified in triplicate PCR reactions using the F515and R805primers designed to be universal for bacterial and archaeal taxa(Bates et al.,2011;Bergmann et al., 2011).Here,we only report results for the bacterial commu-nity as archaea were rare(typically less than1%of16S rRNA sequences).Amplicons were pooled and equimolar concentra-tions pyrosequenced at EnGenCore(University of South Caro-lina)on a Roche FLX454automated pyrosequencer running the Titanium chemistry.Processing of raw bacterial sequence data was performed using QIIME(Caporaso et al.,2010). Briefly,quality sequence reads were grouped by their unique barcode and assigned to phylotypes(!97%similarity),repre-sentative sequences were aligned using PyNAST against the Greengenes core set(DeSantis et al.,2006)and taxonomy was assigned using the Ribosomal Database Project classifier (Wang et al.,2007).A phylogenetic tree containing the aligned sequences was then produced using FastTree(Price et al., 2009).All downstream analyses were determined after sam-ples were rarified to200sequences/sample(to include the maximum number of samples),but the patterns were nearly identical to those observed with1000sequences/sample(data not shown).Relatedness of bacterial communities between each pair of samples(ambient vs.N treatment)was deter-mined using the unweighted Unifrac algorithm,hereon referred to as‘pairwise Unifrac’(Lozupone&Knight,2005). Of the28soils,DNA was successfully amplified from26soils and used for subsequent analyses(GCE and SJQ were excluded).Enzyme activity and temperature sensitivityTo understand the response of microbial carbon and nutrient breakdown activity to N addition across soils,we measured potential extracellular enzyme activity(EEA)for eight extra-cellular enzymes in all28soils:b-1,4-glucosidase(BG)and cel-lobiohydrolase(CBH),b-N-acetylglucosaminidase(NAG), leucine aminopeptidase(LAP),phosphatases(AP)and peroxi-dase(PER)and phenol oxidase(POX)(Table3).Following Saiya-Cork et al.(2002),soil was suspended in sodium acetate (pH5.0)or sodium bicarbonate(pH8.0)buffer and aliquots were dispensed into96-well microplates with16replicate wells per sample per assay.Blank,negative control,and quench wells each had eight replicates.After incubation(20°C, for up to4h depending on substrate),fluorescence was mea-sured using365nm excitation and450nm emissionfilters. Phenol oxidase and peroxidase activities were measured spectrophotometrically(450nm)using L-3,4-dihydroxyphe-nylala-nine(DOPA)(Sinsabaugh et al.,2005).All EEAs were log-transformed to normalize distributions.In addition to comparing the response of individual EEA levels to N addition, we also examined ratios of EEAs to identify relative shifts in nutrient acquisition activities:ln(BG):ln(NAG+LAP)(C:N Fig.1Map of the28soil collection sites.©2012Blackwell Publishing Ltd,Global Change Biology,18,1918–1927 1920K.S.RAMIREZ et al.T a b l e 1D e s c r i p t i o n o f t h e 28c o l l e c t i o n s i t e s a n d t h e s o i l s u s e d i n t h i s s t u d yS i t e A b b r e v i a t i o nE c o s y s t e mL a t i t u d e L o n g i t u d e M A T (°C )M A P (c m )S o i l p H W H C C (m g g À1)N (m g g À1)C :N S a n d S i l tC l a yA m e r i c a n P r a i r i e A P P r a i r i e /G r a s s l a n d 47.80À107.956.227.27.290.68222.110.4847.52527.5A n d r e w s L T E R A N D C o n i f e r o u s F o r e s t 44.20À122.207.8147.05.581.0242.1221.0582.5152.5A r c t i c L T E R A R C A r c t i c T u n d r a 68.63À149.60À10.317.35.470.4522.91.515.27403525B a l t i m o r e E c o s y s t e m S t u d y B E SU r b a n -t e m p e r a t e39.61À76.7413.689.45.150.9284.9421.2377.5157.5B o n a n z aC r e e k L T E R B N Z B o r e a l F o r e s t 64.77À148.27À3.724.44.990.8228.91.7175042.57.5C e d a r C r e e k N a t u r a l H i s t o r y A r e a CD RG r a s s l a n d45.40À93.205.960.76.220.409.91.37.6287.5102.5C o w e e t a L T E R C O WD e c i d u o u s F o r e s t 35.0383.4311.1164.24.630.9459.11.636.9477.51012.5F l o r i d a C o a s t a lE v e r g l a d e s L T E RF C EC o a s t a l25.56À80.3923.6119.07.520.4321.20.730.2985105G e o r g i a C o a s t a l E c o s y s t e m s G C EC o a s t a l31.40À81.3019.4106.04.240.5729.91.421.36952.52.5G u a n i c a G U A D r y f o r e s t 17.97À65.5024.2162.67.810.9668.66.99.94652015H a r v a r d F o r e s t L T E R H F R D e c i d u o u s F o r e s t 42.30À72.108.194.14.451.1062.13.716.7882.512.55H u b b a r d B r o o k E x p e r i m e n t a l F o r e s t H BD e c i d u o u s F o r e s t43.96À71.725.587.04.371.4961.83.816.2677.5157.5I t a s c a S t a t e P a r k I T A D e c i d u o u s F o r e s t 47.27À95.333.065.06.951.0959.34.712.627517.57.5J e p s o n P r a i r i e J E P P r a i r i e -G r a s s l a n d 38.35À121.8215.749.75.920.4413.9113.9454015J o r n a d a B a s i n L T E R J R N D e s e r t 32.49À106.7814.522.97.280.331.8n /a n /a 8012.57.5K a n k a n k e e S a n d s K A N P r a i r i e -G r a s s l a n d 41.05À87.469.977.26.730.5914.51.113.1857.532.510K e l l o g g B i o l o g i c a l S t a t i o n L T E R K B SG r a s s l a n d42.40À85.409.276.26.510.8035.22.812.577027.52.5K o n z a P r a i r i e B i o l o g i c a l S t a t i o n K N ZP r a i r i e -G r a s s l a n d39.08À96.5712.572.46.930.9942.83.512.2342.54017.5L u q u i l l o L U Q T r o p i c a l f o r e s t 18.31À65.7625.7157.55.081.3629.12.312.6537.542.520N i w o t R i d g e N I W A l p i n e T u n d r a 40.09À105.663.939.05.350.7041.41.429.57602515O l y m p i c N a t i o n a l P a r k O L YC o n i f e r o u s F o r e s t47.86À124.045.0300.04.631.0065.12.823.2580155O r d w a y -S w i s h e r O R D W e t l a n d 29.68À82.0020.5110.65.870.326.20.415.5952.52.5S a n J o a q u i n E x p e r i m e n t a l R a n g e S J QD r y S h r u b l a n d37.06À95.6814.959.76.450.4710.70.715.2982.512.55S a n t a R i t a E x p e r i m e n t a l R a n g e S R ED e s e r t31.83À110.8519.127.46.010.292.80.12880155S e v i l l e t a L T E R S E V D e s e r t 34.35À106.8812.919.68.290.322.20.21182.5107.5S h o r t G r a s s S t e p p e S G S P r a i r i e -G r a s s l a n d 40.81À104.197.932.35.930.489.10.811.386522.512.5W a l k e r B r a n c h W B D e c i d u o u s -F o r e s t 35.9784.2813.1117.46.010.7837.81.919.8947.537.515W i n d R i v e rW RC o n i f e r o u s -F o r e s t 45.82121.957.7179.45.60.7732.71.620.4482.5107.5©2012Blackwell Publishing Ltd,Global Change Biology ,18,1918–1927CONSISTENT NITROGEN EFFECTS ON SOILS ACROSS ECOSYSTEMS 1921acquisition);ln BG:ln(AP)(C :P acquisition);and ln(LAP +NAG):ln(AP)(N :P acquisition)(Sinsabaugh et al.,2009).To determine responses of temperature sensitivity to N additions,five replicates for each soil were sealed with a cap containing a rubber septum,and then distributed over five temperatures (10,15,20,25and 30°C).These soils were incu-bated for 18–72h and then respiration rates were determined as described above.This process was repeated 15times over 365days.We used the Arrhenius equation to calculate the apparent activation energy of the chemical reactions that con-tributed to respiration (Ea)where the rate of respiration is rel-ative to total SOC (Laidler,1984).Specifically,Ea was calculated as the slope of the relationship between À1/RT and the natural logarithm of relative respiration rates to yield an index of the relative recalcitrance of the SOC pools mineral-ized by microbes with and without added N (Craine et al.,2010).Statistical analysisThe differences between ambient and treatment pairs in the responses of soil respiration,microbial biomass,the relative abundances of bacterial groups,enzyme activity and tempera-ture sensitivity were assessed with paired t -tests.Our goal was to assess responses across the entire soil collection,rather than isolate differences between individual pairs of sites,thus our experimental design did not rely on replicated ambient and treatment pairs of each soil type.identify significantchanges within the whole bacterial community from ambient to treatment samples,we used a one-sample t -test on pairwise Unifrac values.Bivariate relationships were measured with Pearson correlations and regression analysis (temperature sen-sitivity),with each sample from one of the 28sites represent-ing an individual data point as our goal was to assess responses of the entire soil collection,rather than isolate dif-ferences between individual pairs of sites.All statistical analy-ses were performed using the program R version 2.13.1(The R Foundation for Statistical Computing 2011).ResultsNitrogen effects on soil respiration and microbial biomassAcross the 28soils,N addition tended to suppress soil respiration and microbial biomass.N addition decreased soil respiration for 75%of the soils and decreased microbial biomass for 89%of the soils (Fig.2a,b and Table 2,Table S1).On average,the N addition decreased soil respiration by 11.8%and microbial biomass by 35.2%(P <0.05,and <0.001respectively)but the magnitude of the N response ranged across the soils (from À44.2%to 30.4%for soil respiration,and À86.7%to 36.8%for microbial bio-mass)(Fig.2c).N addition reduced respiration the most for soils with lower soil N content (r =0.41,P =0.04)and higher available C (r =À0.63,P <0.001)N A m e n d e d R e s p i r a t i o n (µg C g C –1 h –1)Ambient Respiration (µg C g C –1 h –1)Ambient Biomass (µg C g C –1 h –1)N A m e n d e d B i o m a s s (µg C g C –1 h –1)0100200300400500100200300400500(a)(b)(c)2040608010020406080100% C h a n g e f r o m a m b i e n t s o i l s–80–60–40–2002040Respiration BiomassFig.2Ambient (no N added)soil respiration plotted against N amended soil respiration (a)and ambient (no N added)microbial bio-mass plotted against N amended microbial biomass (b),where points below (gray)the dashed line indicate a decrease with the N amendment.Boxplot summarizing relative change in respiration and biomass with the N amendment (c);the solid black line represents the mean of all soils.Table 2Pearson correlation coefficients relating microbial and soil changes to edaphic factorsNC CN Available C pH Latitude MAT MAP TextureD Respiration 0.41*0.15À0.34À0.62**0.22À0.090.340.3À0.26D Biomass0.310.170.01À0.23À0.190.360.05À0.080.1Pairwise Unifrac À0.52**À0.50**0.080.310.08À0.19À0.17À0.340.1*P <0.05;**P <0.01.©2012Blackwell Publishing Ltd,Global Change Biology ,18,1918–19271922K.S.RAMIREZ et al.(Table2,Fig.3a,b).Similarly,N addition tended to cause the largest reductions in microbial biomass in soils with low N content(r=0.32,P=0.10)(Table2, Fig.3c).Bacterial community responses to nitrogen amendments Of the26soils that were successfully sequenced,N addition had consistent effects on bacterial community composition regardless of community starting point (Table S2).Overall pairwise Unifrac distances between ambient and N treatment samples indicates that,on average,bacterial communities significantly shifted with the N treatment(P<0.001,one sample t-test).N addition induced the most pronounced shifts in overall bacterial community composition in those soils with lower C and N concentrations(Table2).More specifi-cally,N addition increased the relative abundance of Actinobacteria and Firmicutes by an average of11.8% and2%,respectively,while decreasing the abundance offive phyla:Acidobacteria(À13.5%),Verrucomicrobia (À5%),Cyanobacteri a(À1%),Planctomycetes(À1%)and Deltaproteobacteria(À1%)(Fig.4,Table S3)(all P<0.05).Unlike whole community shifts,the change in relative abundance of individual phyla between ambient and treatment samples could not be predicted by initial soil C or N concentrations(P>0.05in all comparisons).Enzyme activities and temperature sensitivitySimilar to the declines in soil respiration and microbial biomass,N addition also decreased the potential activi-ties of four extracellular enzymes:b-1,4-glucosidase (À11.6%),acid phosphatase(À15.1%),leucine amino peptidase(À71.1%)and peroxidase activity(À24.7%) (all P<0.001)(Table3,Table S4).The other three extra-cellular enzymes also exhibited N-induced declines in activities,but the changes were not significant.N addi-tion did not alter the ratios of potential extracellular©2012Blackwell Publishing Ltd,Global Change Biology,18,1918–1927CONSISTENT NITROGEN EFFECTS ON SOILS ACROSS ECOSYSTEMS1923enzyme activities that would indicate a change in nutri-ent requirements(Table3),instead suggesting that the decline in enzyme activities are non-selective and sim-ply associated with the decreases in microbial biomass with N.Unlike soil respiration and microbial biomass, the magnitude of change in enzyme activities was not associated with variation among soils in total N,C or available C(P>0.05for all comparisons,Table S5)as we observed with soil respiration and microbial bio-mass.Across the incubation period,N-amended microbial communities were degrading C with lower biochemical recalcitrance than ambient communities,as determined by the average decrease in activation energy(E a)from 85.7to83.2kJ molÀ1(P=0.002).The effect of N on the temperature sensitivity of microbial decomposition did not change linearly over time,but did differ between sites(Table4).Sites with an intermediate pH(5.5) had the lowest decline in E a with N addition,while sites with extreme pH had the greatest declines (dEaN=11.1–1.81*pH– 2.37*(pH-5.88)2;r2=0.38, P=0.003).There were no relationships between the response of E a to N addition with microbial biomass, site climate,or soil characteristics(P>0.05for all com-parisons).DiscussionAcross the wide array of soil types,soil respiration and microbial biomass were consistently suppressed under N amendments by an average of11.8%and35.2%, respectively.Thesefindings correspond with results obtained infield studies,where decreases in soil respi-ration ranged between8%and15%and biomass decreases ranged between11%and20%(Treseder, 2008;Janssens et al.,2010;Liu&Greaver,2010).How-ever,unlike previous studies,this work demonstrates that belowground responses to N enrichment are not solely related to changes in plant C inputs to soil,as the soils in this study did not receive any C inputs during the course of this incubation.Admittedly,by eliminat-ing plant C inputs,we are choosing to focus on the direct effects of N alone and ignoring indirect N effects that likely contribute to changes in soil communities and processes observed infield experiments.In the field,N amendments generally increase aboveground primary productivity and thereforefield studies cannot fully separate the direct effects of N from indirect increases in the quantity or quantity of plant C inputs to soil.In contrast,our lab incubation demonstrates that microbial activities,biomass,and community composi-tion can change independently of alterations in litter quality and C inputs,and were most likely a direct response to increased N.Therefore,to better identify the mechanisms driving the suppression of decomposi-tion,we combined the measurements of microbial res-piration and biomass with analyses of bacterial community structure,extracellular enzyme activities and temperature sensitivity.Sequencing analysis revealed that with the N amend-ment there were consistent phylum-level changes in the bacterial communities across soils.Generally,Actinobac-teria and Firmicutes increased in abundance,and Acido-bacteria and Verrucomicrobia decreased in abundance. Thesefindings are similar to previousfield studies, where comparable increases and decreases were observed in the same bacterial groups(Nemergut et al., 2008;Campbell et al.,2010;Ramirez et al.,2010b).Shifts in bacterial phyla may be a function of changes in rela-tive abundances atfiner levels of taxonomic resolution, but for this study higher resolution comparisons were not appropriate as all soils had such different starting communities,with little overlap at the order or class levels.Overall,the changes observed here and in other studies,support the copiotrophic hypothesis.This hypothe-sis predicts that under N amendments,microbial groupsTable3Mean enzyme activities and significance values from paired t-tests comparing ambient to samples receiving added NEnzyme EC Mean relative change in activity(%)Pb-1,4-glucosidase 3.2.1.21À23.6<0.001Cellobiohydrolase 3.2.1.91À11.40.21b-N-acetylglucosaminidase 3.2.1.14À0.210.98Leucyl aminopeptidase 3.4.11.1À64.1<0.001Acid phosphatase 3.1.3.2À36.0<0.001Phenol oxodiase 1.10.3.2À15.40.06Peroxidase 1.11.1.7À24.9<0.001BG:AP–17.30.20BG:(LAP+NAG)–0.910.98(LAP+NAG):AP– 4.990.80EC,enzyme commission number.Table4Regression results for E a measured across15timepoints during a1-year incubation as a function of site identity,N treatment,time into the incubation,and all pairwise interac-tionsSum of squares PSite132196<0.001N13520.002Time11213<0.001Site*N7760<0.001Site*Time13407<0.001N*Time310.64©2012Blackwell Publishing Ltd,Global Change Biology,18,1918–1927 1924K.S.RAMIREZ et al.。