Submitted 9 November 2018Accepted 24 June 2019Published 17 July 2019

Corresponding authorsXiaoqin Chengcxq_200074163comHairong Han hanhr6015bjfueducn

Academic editorJorge Curiel Yuste

Additional Information andDeclarations can be found onpage 16

DOI 107717peerj7343

Copyright2019 Wu et al

Distributed underCreative Commons CC-BY 40

OPEN ACCESS

Microbial regulation of soil carbonproperties under nitrogen addition andplant inputs removalRan Wu Xiaoqin Cheng Wensong Zhou and Hairong HanBeijing Key Laboratory of Forest Resources and Ecosystem Processes Beijing Forestry University BeijingChina

ABSTRACTBackground Soil microbial communities and their associated enzyme activities playkey roles in carbon cycling in terrestrial ecosystems Soil microbial communities aresensitive to resource availability but the mechanisms of microbial regulation have notbeen thoroughly investigated Here we tested the mechanistic relationships betweenmicrobial responses and multiple interacting resourcesMethods We examined soil carbon properties soil microbial community structureand carbon-related functions under nitrogen addition and plant inputs removal(litter removal (NL) root trench and litter removal (NRL)) in a pure Larix principis-rupprechtii plantation in northern ChinaResults We found that nitrogen addition affected the soil microbial communitystructure and that microbial biomass increased significantly once 100 kg haminus1 aminus1

of nitrogen was added The interactions between nitrogen addition and plant inputsremoval significantly affected soil bacteria and their enzymatic activities (oxidases) TheNL treatment enhanced soil microbial biomass under nitrogen additionWe also foundthat the biomass of gram-negative bacteria and saprotrophic fungi directly affected thesoil microbial functions related to carbon turnover The biomass of gram-negativebacteria and peroxidase activity were key factors controlling soil carbon dynamicsThe interactions between nitrogen addition and plant inputs removal strengthened thecorrelation between the hydrolases and soil carbonConclusions This study showed that nitrogen addition and plant inputs removalcould alter soil enzyme activities and further affect soil carbon turnover via microbialregulation The increase in soil microbial biomass and the microbial regulation ofsoil carbon both need to be considered when developing effective sustainable forestmanagement practices for northern ChinaMoreover further studies are also needed toexactly understand how the complex interaction between the plant and below-groundprocesses affects the soil microbial community structure

Subjects Ecology Microbiology Soil Science ForestryKeywords Soil microorganisms Regulation Soil enzyme Nitrogen addition Soil carbonproperties Plant inputs removal

INTRODUCTIONIn the past century human activity has altered atmospheric nitrogen exchange inforest ecosystems (Deforest et al 2004 Galloway 1998) with global rates of nitrogendeposition having more than doubled (Vitousek et al 1997) Nitrogen is an important

How to cite this article Wu R Cheng X Zhou W Han H 2019 Microbial regulation of soil carbon properties under nitrogen additionand plant inputs removal PeerJ 7e7343 httpdoiorg107717peerj7343

element controlling soil quality plant diversity and the productivity of forest ecosystems(Sophie Kerstin amp Michael 2011) Variation in nitrogen may affect soil carbon propertiesby influencing how soil microbial community structure and its functions drive soilcarbon transformation and turnover (Compton et al 2004) Soil microorganisms act asdecomposers and sensitive indicators of soil quality by regulating key process of soil carboncycling including lignin and cellulose degradation and soil carbon turnover (Lei et al2018 Li et al 2018 Mele amp Crowley 2008 Sun et al 2017)

Soil microbial community structure is sensitive to resource availability Depending onresource availability and supply soil microbial growth can either be stimulated or inhibitedNitrogen addition may alter soil microbial community structure and functions in severalways For example the growth of soil microorganisms can be stimulated by low-levelsof nitrogen addition because nitrogen-limitation can be ameliorated by improving soilnitrogen availability (Zhu et al 2016) Similarly carbon-limitation can be relieved byenhancing aboveground net primary production (NPP) and litter decomposition (Sunet al 2016 Zhang et al 2008) De Deyn Quirk amp Bardgett (2011) found that nitrogenaddition influenced microbial community structure by directly enhancing soil nitrogenavailability as well as by indirectly affecting soil microbial functions related to carbonturnover This was accomplished through the stimulation of specific microbial groupsthat influence the soil carbon process In contrast it has been found that nitrogendeposition negatively affects soil microbial growth composition and function (ZhangChen amp Ruan 2018) Overabundance of nitrogen can cause carbon-limitation due tosubsequent decreases in litter decomposition rate This inhibition of litter decompositionis also caused by decreases in phenol oxidase and by reductions in pH or toxicity byaluminum mobilization (Treseder 2010) Excessive nitrogen can also elicit productionof ligninase from some microorganisms including white rot fungi (Kundu amp Chatterjee2006) Therefore examining of soil microbial responses along a nitrogen addition gradientmay help us to understand how nitrogen influences soil carbon processes via the microbialcommunity structure and functions

The microbial response to shifts in multiple interacting limiting resources is not wellunderstood because few studies have been done (Zhang et al 2015a Zhang et al 2015b)Changes in litter input can help improve soil nutrient availability by alleviating resourcestresses (Li et al 2015a Li et al 2015b) Soil carbon processes carried out by soil microbialcommunities rely on complex interactions with carbon input which in turn are mediatedby nitrogen availability (Bloom Chapin amp Mooney 1985 Bohlen et al 2001 Fisk amp Fahey2001) The response of soil microbes to nitrogen addition is further mediated by carboninput changes due to shifts between carbon-limitation and nitrogen-limitation Differencesin microbial responses may be related to the quality of litter and roots (Liu et al 2014)Soil microbial functions relating to carbon acquisition under nitrogen addition may alsodepend on the plant litter and roots (Alster et al 2013)

A better understanding of how carbon availability specifically affects feedback betweenplants andmicroorganisms requires further research on soilmicrobial community structureand functions relative to different plant inputs removal scenarios Zhao et al (2017)conducted a litter removal experiment to study soil microbial community structure and

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function in a managed pine forest However it still remains unclear whether microbialcommunity shifts are a direct result of nitrogen addition or of the carbon inputs that aremediated by nitrogen addition (Li et al 2015a) Previous work on nitrogen addition andplant inputs removal have suggested both positive and negative effects on soil microbialcommunity structure and functions (Bachar et al 2010 Shi et al 2016 Li et al 2015b)Therefore the interaction between nitrogen and changes in carbon input needs to beconsidered furtherwhen analyzingmicrobialmechanisms under different nitrogen additionscenarios (Georgiou et al 2017 Zhang et al 2015a Zhang et al 2015b)

Microbial regulation of soil carbon is still not well understood Ma et al (2018) hasexplained the effect of nitrogen addition on soil total organic carbon and active carboncomponents However in this present study soil carbon properties were used to furtheranalyze the microbial regulation of soil carbon regulatory paths Studying the direct andindirect effects of both microbial community structure on various functions and microbeson soil carbon properties is necessary to better understand the belowground processesaffecting carbon dynamics For example specific microbial groups have certain functionsused to drive soil carbon decomposition and turnover (Wang et al 2017) Likewisesoil enzyme activities are direct expression of soil microbial functions relating to soilcarbon dynamics (Wang et al 2015) These interactions can be divided into oxidases andhydrolases activities (Bissett et al 2011 You et al 2014) Specifically complex compoundslike lignin are degraded by oxidases which are produced primarily by fungi Cellulose isdegraded by hydrolases which are produced primarily by bacteria and relate to soil carbonacquisition (You et al 2014)

The experimental approach of the Detritus Input and Removal Treatment (DIRT)experiments is an efficient method for investigating the correlation between plant and soilmicrobial communities through litter removal and root exclusion treatments (Veres et al2015) Likewise litter removal and root exclusion regulate soil microclimate by influencingevaporation and absorption of precipitation (Fekete et al 2016) Larix principis-rupprechtiis a common plantation tree species in the warm temperate Taiyue Mountains of Shanxiprovince In this study a litter and root exclusion experiment designed in themethod of theDIRT project and the treatments of variable nitrogen addition were established to examinefeedback among plant litter soil nutrient availability and soil microbial communitiesin short-term treatment in L principis-rupprechti plantations We measured hydrolaseactivities to assess soil microbial functions relating to cellulose and chitin degradingcapacity We also assessed oxidase activities to assess microbial functions relating to lignindegradation and their influence on soil carbon transformation We analyzed how shiftsin soil microbial community and functions drive variation in soil carbon propertiesFurthermore we investigated the direct and indirect effects of soil microbial communitieson soil enzyme activities and the direct and indirect effects of microbes on soil carbonproperties under various nitrogen addition and plant inputs removal treatments Theobjectives of this study were (1) to assess the responses of soil microbial communitystructure and functions to nitrogen addition and plant inputs removal and (2) toexplore how nitrogen addition drives linkages among microbial community structurefunctions and soil carbon properties under different plant inputs removal treatments We

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hypothesized that nitrogen addition would change soil microbial community structure andfunctions with and without plant inputs removal and that the soil microbial communitieswould regulate soil carbon properties controlled by microbial functions

MATERIAL AND METHODSStudy sitesStudy sites were located in Larix principis-rupprechtii plantations in the Taiyue Mountain(3635primendash3653primeN 11191primendash11204primeE) of Shanxi province in northern China A warmtemperate and continental monsoon climate is characteristic of the site Its altitude rangesfrom 2100 to 2400 m with an average annual temperature around 86 C The growingseason starts in April and lasts until October and a rainy season is observed from Juneto August Average annual precipitation is nearly 600 mm The predominant soil typethroughout the study sites is Haplic luvisol The dominant tree species are Larix principisndashrupprechtii and Betula platyphylla which are typical temperate tree species of northernChina Lonicera japonica Corylus mandshurica Rubus corchorifolius Rosa xanthina andLespedeza bicolor are common shrub species encountered in the region

Experimental design and treatmentsThe experiment began in August 2014 in a 34-year-old Larix principis-rupprechtiiplantation Plots were established using a nested two-factor design Three replicateplots of two m times two m were established for each treatment These plots were randomlyplaced with a minimum distance of 05 m between plots For the treatment we used fournitrogen addition levels and three plant inputs removal treatments which were nestedwithin each nitrogen addition level The three treatments of plant inputs removal includedroot trenching and litter removal (NRL) just litter removal (NL) and a control plot (C)where litter and roots were left intact In the NRL treatment living roots and abovegroundlitter were excluded and trenches were excavated to a depth of 05 m This was doneto restrict roots entering the plots from nearby trees Trenches were embedded withasbestos shingles and then refilled with soil In the NL treatment all aboveground inputswere removed The aboveground litter was removed monthly during the growing seasonNatural above- and below-ground litter inputs were left in the C treatment The nitrogenaddition treatments were N0 (0 kg haminus1 aminus1) N1 (50 kg haminus1 aminus1) N2 (100 kg haminus1 aminus1)and N3 (150 kg haminus1 aminus1) NH4NO3 was used for the nitrogen addition treatments whichwere conducted once a month during the growing season from 2014 to 2016

Soil samples were collected in August 2015 and 2016 at five random points in eachplot at a depth of 0ndash10 cm A metal corer with an inner diameter of five cm was used tocollect samples in the field The five soil samples from a single plot were mixed to createa composite sample Soil samples were then stored in sealed bags and immediately takenback to the laboratory for analysis A two mm sieve was used to remove gravel roots andlarge organic residues from the samples Samples were then used to measure soil microbialcommunity structure soil enzyme activities and soil carbon properties This data was usedto assess correlations among the samples

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Analyses of soil propertiesSoil organic carbon (SOC) was analyzed with an elemental analyzer (FLASH 2000) Soilmicrobial biomass carbon (MBC) was determined via the fumigation-extraction methodand each sample was fumigated for 24 h at 25 C with alcohol-free CHCl3 Dissolvedorganic carbon (DOC) and MBC were measured using a 05 M K2SO4 extracting agentand measured with a Multi NC 3100 TOC analyzer (Chen et al 2017) Soil microbialcommunity structure was determined using phospholipid fatty acid (PLFA) analysisPLFAs were calculated based on a 190 internal standard content After addition of aninternal standard the phospholipid fraction was subjected to a mild alkaline methanolysisand the resulting fatty acid methyl esters were separated on a gas chromatograph (Bossio ampScow 1998) The following soil microbial groups were classified using diagnostic fatty acidsas indicators gram-positive bacteria (GP) gram-negative bacteria (GN) Saprotrophicfungi (Sap) arbuscular mycorrhizal fungi (AMF) and actinomycete (Act) (Table S1) Theactivities of phenol oxidase (PO) and peroxidase (PER) were determined using DOPA (3 4-Dihydroxy- L- phenylalamine) as the substrate Soil suspensions (ie one g fresh soil with1 five mL 50 mmol Lminus1 sodium acetate buffer) and two mL five mmol Lminus1 L-DOPA weremixed for the phenol oxidase assay The same suspension was used for peroxidase analyseswith an addition of 0 two mL 03 H2O2 (Baldrian 2009) The activities of β-glucosidase(BG) N-acetyl-β-glucosaminidase (NAG) and cellobiohydrolase (CBH) were measuredwith p-nitrophenol assays (You et al 2014)

StatisticsAnalysis of variance (ANOVA) was used to assess variation in soil carbon soil microbialcommunities and soil enzyme activities under various nitrogen addition and plant inputsremoval treatments Statistical analyses were performed using SPSS 190

Redundancy analysis (RDA) was used to assess linkages between soil microbialcomposition soil enzyme activity and soil carbon properties RDA was used with forwardselection to filter the relative importance of individual explanatory variables and to predictthe variation in soil microbial community structure enzyme activity and soil carbonunder nitrogen addition both with and without plant inputs removal These analyses werecompleted in CANOCO software for Windows 45

Lastly structural equation modelling (SEM) was used to test the hypothesis that soilmicrobial communities affect soil carbon properties by influencing soil enzyme activitiesthrough the combined effects of nitrogen addition and plant inputs removal Path modelswere established based on existing literature where bacteria and Sap represent the structuralattributes of soil microbial communities and oxidases and BG represents the soil microbialfunction driving carbon acquisition and oxidation (You et al 2014) We combined thedata from all treatments and estimated the model parameters using a maximum likelihoodestimation in the Amos 220 software package The adequacy of model fit was assessed by aχ2 test (p gt 005 CMINdf lt 2) comparative fit index (CFI gt 090) and by the root squaremean error of approximation (RMSEA lt 005) (You et al 2014) Numbers on arrows arestandardized direct path coefficients R2 values represent the proportion of total varianceexplained for a specific dependent variable Dash-line arrows indicate negative effects

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Figure 1 Soil microbial biomass under nitrogen addition in the NRL (AndashB) NL (CndashD) and C (EndashF)treatmentsGP Gram-positive bacteria GN Gram-negative bacteria AMF arbuscular mycorrhizal fungiSap Saprotrophic fungi Act actinomycete NRL root trench and remove litter NL litter removal C thecontrol plot N0 nitrogen addition at 0 kg haminus1 aminus1 N1 nitrogen addition at 50 kg haminus1 aminus1 N2 nitro-gen addition at 100 kg haminus1 aminus1 N3 nitrogen addition at 150 kg haminus1 aminus1 indicates that the microbialbiomass differs significantly from the N0 treatment at each nitrogen level (plt 005)

Full-size DOI 107717peerj7343fig-1

RESULTSVariation in soil microbial biomass under nitrogen additionIn the C treatment soil microbial biomass first increased and then decreased withan increase in nitrogen addition level (Fig 1) In the NL treatment nitrogen additionenhanced soil microbial biomass The increase in soil microbial biomass was significantlyhigher in the N1 level treatment than at other nitrogen levels in 2016 In the NRL treatmentsoil microbial biomass increased under nitrogen addition in the first year but decreasedunder the same treatment in 2016

Soil microbial biomass was significantly influenced by sampling year (Table 1) Therewere also significant interactive effects between plant inputs removal and sampling year

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Table 1 Result (F value) of a threemdashway ANOVA on the effects of nitrogen addition plant inputs re-moval and year on soil microbial biomass

Treatments GP GN AMF Sap Act

N 7673 1863 6644 8020 1117L 34228 6409 5641 4524 1643Y 225004 1144864 511431 15818 171889

N Y 1365 0117 0606 0975 3473

L Y 10023 10483 0046 0442 8476

N L 3027 2612 1131 2237 3030N L Y 2814 3151 1339 0690 1705

NotesGP Gram-positive bacteria GN Gram-negative bacteria AMF arbuscular mycorrhizal fungi Sap Saprotrophic fungiAct actinomycete N nitrogen addition L plant inputs removal Y yearEach of these variables were fitted in following statistical model eg four levels of nitrogen addition three plant inputs treat-ments two yearsp lt 005p lt 001plt 0001

on the biomass of GP GN and Act Interactive effects were also found between nitrogenaddition and plant inputs removal on the biomass of GP and GN though the effectsdiffered with year

Variation in soil enzyme activity under nitrogen additionIn the C treatment in 2015 nitrogen addition enhanced the activities of PO PER BG andNAG (Fig 2) In contrast the activity of PO had a decreased trend in 2016 In the NL andNRL treatment PO and PER activities increased under nitrogen addition in 2015 but theydecreased in 2016 Moreover the N1 level treatment enhanced the activity of NAG in theC and NL treatments

Nitrogen addition plant inputs removal and sampling year all significantly affected soilenzyme activities (Table 2) The effect of plant inputs removal or nitrogen addition onthe activities of PER NAG and CBH were also dependent on year There were interactiveeffects between nitrogen addition and plant inputs removal on the activities of PER andBG in all years

Variation in soil carbon properties under nitrogen additionIn the C treatment in 2015 the N1 and N2 level treatments lowered SOC and DOCHowever there was no significant variation in SOC and DOC in the N1 and N2 leveltreatments in 2016 (Table 3) In the NL treatment in 2015 SOC and MBC first increasedand then declined with nitrogen addition DOC declined gradually with nitrogen additionin 2015 In the NRL treatment in 2015 SOC had a trend of decrease at the N3 levels At theN2 level treatment MBC was significantly higher than at the other nitrogen levels in 2015but the opposite was true in 2016 DOC had a trend of decrease under nitrogen additionin the NRL treatment during both sampling years

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Figure 2 Soil enzyme activities under nitrogen addition in the NRL (AndashB) NL (C-D) and C (E-F) treat-ments PO phenol oxidase PER peroxidase BG β-glucosidase NAG N-acetyl-β-glucosidase CBH cel-lobiohydrolase NRL root trench and remove litter NL litter removal C the control plot N0 nitrogenaddition at 0 kg haminus1 aminus1 N1 nitrogen addition at 50 kg haminus1 aminus1 N2 nitrogen addition at 100 kg haminus1

aminus1 N3 nitrogen addition at 150 kg haminus1 aminus1 indicates that soil enzyme activities differs significantlyfrom the N0 treatment at each nitrogen level (plt 005)

Full-size DOI 107717peerj7343fig-2

Microbial regulatory pathways on soil enzymes undernitrogen additionThe pathway showed that GN and Sap had a direct and indirect effect on soil enzymeactivities under nitrogen addition (Table 4) The biomass of GN had relatively strong totaleffects on the soil enzyme activities For the C treatment the biomass of GN had a directand positive effect on the activities of PER and BG while Sap had a direct effect on theactivity of PER In the NL and NRL treatments the activities of PER and BG were directlyaffected by the biomass of GN Compared to the total effects of plant inputs removal

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Table 2 Result (F value) of a threemdashway ANOVA on the effects of nitrogen addition plant inputs re-moval and year on soil enzyme activities

Treatments PO PER BG NAG CBH

N 1066 10504 16875 20183 13420

L 33173 111121 79646 54570 72586

Y 215231 382211 165531 97786 22437

N Y 6979 19828 6987 6723 6003

L Y 1944 25067 2482 13460 16278

N L 2723 14129 12178 1189 6613

N L Y 1699 3936 4254 1592 1922

NotesPO phenol oxidase PER peroxidase BG β-glucosidase NAG N-acetyl-β-glucosidase CBH cellobiohydrolase N ni-trogen addition L plant inputs removal Y yearEach of these variables were fitted in following statistical model eg four levels of nitrogen addition three plant inputs treat-ments two yearsplt 005plt 001plt 0001

Table 3 The variation in soil carbon properties under nitrogen addition in the NRL NL and C treatments

Soil carbon Plant inputsremoval

Years The levels of nitrogen addition

N0 N1 N2 N3

NRL 2015 5345plusmn 742a 5527plusmn 1337a 527plusmn 352a 4013plusmn 61aNL 426plusmn 642b 656plusmn 843a 467plusmn 285b 4065plusmn 035bC 5123plusmn 757a 4437plusmn 372ab 3997plusmn 101b 377plusmn 601bNRL 2016 4163plusmn 343a 3785plusmn 251a 377plusmn 173a 4119plusmn 223aNL 4236plusmn 191a 4513plusmn 44a 4071plusmn 137a 3996plusmn 316a

SOC (g kgminus1)

C 4504plusmn 467ab 5057plusmn 094a 4914plusmn 171a 4333plusmn 35bNRL 2015 149008plusmn 4936c 111513plusmn 6448d 202212plusmn 2669a 167574plusmn 453bNL 128753plusmn 191d 216646plusmn 2779a 17986plusmn 4693b 158109plusmn 12484cC 149285plusmn 3398b 14748plusmn 3883b 142357plusmn 8389b 167182plusmn 4868aNRL 2016 90213plusmn 8186a 88561plusmn 8273a 69232plusmn 30633a 88768plusmn 4931aNL 102238plusmn 15233a 8227plusmn 15768a 98509plusmn 5154a 92088plusmn 1091a

MBC (mg kgminus1)

C 106401plusmn 1242a 9346plusmn 5231a 103153plusmn 15929a 98817plusmn 2851aNRL 2015 13825plusmn 2584a 13339plusmn 1251a 13507plusmn 007a 12625plusmn 242aNL 14002plusmn 294a 13252plusmn 1628ab 12519plusmn 476ab 11501plusmn 66bC 1939plusmn 374a 1653plusmn 693b 15963plusmn 2377b 1815plusmn 242abNRL 2016 1735plusmn 824a 16159plusmn 1026a 15472plusmn 1075b 15867plusmn 1002abNL 16502plusmn 788a 15919plusmn 731a 15993plusmn 1039a 16278plusmn 1569a

DOC (mg kgminus1)

C 17992plusmn 689a 17951plusmn 1625a 19183plusmn 914a 17369plusmn 1321a

NotesDifferent letters indicate significant differences under different nitrogen addition (plt 005) Values are meanplusmn standard errors (n= 3)SOC soil organic carbon MBC soil microbial biomass carbon DOC dissolved organic carbon NRL root trench and remove litter NL litter removal C the controlplot N0 zero rate of nitrogen addition N1 nitrogen addition at 50 kg haminus1 aminus1 N2 nitrogen addition at 100 kg haminus1 aminus1 N3 nitrogen addition at 150 kg haminus1 aminus1

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Table 4 Path coefficients of the effects of soil microbes on soil enzyme activities under nitrogen addi-tion in the NRL NL and C treatments

Factors Effect Soil enzyme

PER BG

In the NRL treatmentDirect 0716 0733Indirect 0 0GN

Total 0716 0733Direct 0 0Indirect 0204 0210Sap

Total 0204 0210

In the NL treatmentDirect 0844 0749Indirect 0 0GN

Total 0844 0749Direct 0 0Indirect 0384 0341Sap

Total 0384 0341

In the C treatmentDirect 0635 0526Indirect 0091 0GN

Total 0726 0526Direct 0329 0Indirect 0177 0146Sap

Total 0506 0146

NotesPER peroxidase BG β-glucosidase GN Gram-negative bacteria Sap Saprotrophic fungi NRL root trench and removelitter NL litter removal C the control plot

we found that the total effect of GN augmented the activity of PER in the NL and NRLtreatments while the total effect of Sap reduced the activity of PER The total effects of GNand Sap increased the activity of BG in the NL and NRL treatments

Soil enzyme regulatory pathways on soil carbon properties undernitrogen additionThe pathway showed that soil enzymes had both direct and indirect effects on soil carbonunder nitrogen addition (Table 5) The activity of PER had relatively strong total effects onsoil carbon In the C treatment SOC was directly and negatively affected by the activity ofPER while MBC was directly and positively influenced by PER activity There was no directeffect of BG activity on soil carbon properties In the NL treatment the activities of PERand BG directly affected MBC but only PER directly affected SOC In the NRL treatmentMBC was directly affected by BG activity However PER and BG did not directly influenceSOC Compared to the total effects under nitrogen addition and plant inputs removal wefound that the total effect of PER reduced MBC and SOC in the NRL treatments The totaleffect of BG augmented MBC and SOC in the NL treatments

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Table 5 Path coefficients of the effects of soil enzyme activities on soil carbon under nitrogen additionin the NRL NL and C treatments

Factors Effect Soil carbon

MBC SOC

In the NRL treatmentDirect 0 0Indirect 0437 0PER

Total 0437 0Direct 0691 0Indirect 0 0BG

Total 0691 0

In the NL treatmentDirect 0556 0479Indirect 0305 0PER

Total 0861 0479Direct 0399 0Indirect 0425 0366BG

Total 0824 0366

In the C treatmentDirect 0825 minus0495Indirect 0 0PER

Total 0825 minus0495Direct 0 0Indirect 0305 minus0183BG

Total 0305 minus0183

NotesPER peroxidase BG β-glucosidase MBC soil microbial biomass carbon SOC soil organic carbon NRL root trenchand remove litter NL litter removal C the control plot

Regulation of soil carbon by the soil microbial communities andenzymes under nitrogen additionAmong soil microbial community and soil enzyme variables RDA ordination indicatedthat the biomass of GN and the activity of PER significantly affected soil carbon propertiesunder nitrogen addition (Table 6) The path of the variables under the interactionbetween nitrogen addition and plant inputs removal passed the statistical test for adequacy(χ2= 4468 p = 0614 CNMIdf = 0745 CFI = 1000 GFI = 0979 RMSEA lt0001)

and the non-significant pathways were deleted (Fig 3) The model explained 56 and 9of the variance in the activities of PER and BG respectively Likewise 66 and 27 ofthe variance in MBC and SOC were explained by this model The path analysis indicatedthat GN and Sap regulated soil oxidases hydrolases and soil carbon properties undernitrogen addition and plant inputs removal SOC was negatively and directly affected byPER activity which was positively influenced by GN and Sap GN and Sap directly andpositively influenced soil carbon

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Table 6 Marginal and conditional effects of soil microbial composition and soil enzyme activities onsoil carbon obtained from forward selection in Redundancy analysis (RDA) under nitrogen addition

Variables Lambda-Aa Lambda-Bb p c F-ratiod

GN 0515 0515 0002 72223PER 0234 0028 0020 4082

NotesaDescribe marginal effects which show the variance explained when the variable is used as the only factorbDescribe conditional effects which show the additional variance each variable explains when it is included in the modelcThe level of significance corresponding to Lambda-B when performing Monte Carlo test at the 005 significance leveldThe Monte Carlo test statistics corresponding to Lambda-B at the 005 significance levelPER peroxidase GN Gram-negative bacteria

Figure 3 The structural equationmodel depicting the regulation of soil carbon by enzyme activitiesand the soil microbial community under the combined effects of nitrogen addition and plant inputs re-movalNumbers on arrows are standardized direct path coefficients R2 value represents the proportionof total variance explained for the specific dependent variable Dash-line arrows indicate negative effectsPER peroxidase BG β-glucosidase GN Gram-negative bacteria Sap Saprotrophic fungi SOC soil or-ganic carbon MBC soil microbial biomass carbon

Full-size DOI 107717peerj7343fig-3

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DISCUSSIONThe responses of soil microbial communities and functions tonitrogen addition and plant inputs removalAn important finding in our study include that soil microbial biomass increased undernitrogen addition and that microbial biomass was significantly higher in the N2 leveltreatment than the other levels This result is consistent with previous observations showingincreases in soil bacterial biomass and diversity after nitrogen addition (Nguyen et al 2018)as well as increases in bacterial biomass in a lower-elevation forest and in fungal biomassin an upper-elevation forest after nitrogen addition (Cusack et al 2011) This result showsthat the N1 treatment enhanced the activity of NAG which degrades chitin This suggeststhat microbial turnover occurs more rapidly at the N1 level treatment possibly becausemicrobial cell walls consist of fungal chitin and bacterial peptidoglycan Our findingsalso suggest that soil oxidase activities increased after the first year of nitrogen additionand then decreased after the second year Similar results were also observed in an Alaskanboreal forest where carbon-degrading enzyme activities increased after short-term nitrogenaddition (Allison Czimczik amp Treseder 2008) However declines in soil bacterial biomasshave also been reported after three years of nitrogen addition in a secondary tropical forestof China and one year of nitrogen addition in North America (Li et al 2015a Li et al2015b Ramirez Craine amp Fierer 2012) It has also been found that long-term nitrogenaddition reduces soil fungi via nitrogen saturation in temperate ecosystems (DemolingNilsson amp Baringaringth 2008) Our results show that nitrogen addition reduced SOC and DOCand increased soil microbial biomass in the first year of treatment It has been suggestedthat soil microbial biomass correlates negatively with SOC under nitrogen addition Thisresult was supported by the literature showing a negative relationship between soil bacterialbiomass and SOC over three years of nitrogen addition in a temperate needle-broadleavedforest (Cheng Fang amp Yu 2017) Similar results were reported by other studies that founddeclines in soil organic carbon under nitrogen addition in grasslands (Moinet et al 2016)

It has been theorized that changes in soil organic matter may result from variationin soil nutrient availability following plant inputs removal Lietal2015 Our results showthat nitrogen addition lowered soil microbial biomass and soil oxidases activities in theNRL treatment in 2016 The response of soil microbial communities to nitrogen additiondid not align with the response of communities to the plant inputs removal It has beenindicated that plant inputs removal could potentially alter nutrient limitation undernitrogen addition (Sayer et al 2012) Likewise after the first year of root trenching andlitter removal soil microbial biomass and oxidase activities increased under nitrogenaddition because they had been previously nitrogen-limited However these metricsdeclined in the second year due to a shift from nitrogen-limitation to carbon-limitation(Averill amp Waring 2018 Traoreacute et al 2016) Microbial biomass was much higher at theN1 level than at other nitrogen addition levels in the second year of litter removal Thismay have been because litter removal caused nitrogen-limitation and nitrogen additionpositively affected soilmicrobes at theN1 levelWe also found that SOC andDOCdecreasedwith nitrogen addition in the NRL treatments due to the shift in nutrient limitation Our

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result is inconsistent with previous work showing that soil organic matter decompositiondoes not differ under nitrogen addition between root exclusion and natural states This isbecause the presence of roots mediates the response of soil organic carbon decompositionto nitrogen addition (Moinet et al 2016)

Plant inputs removal altered soil bacterial and fungal communities which aligned withprevious literature (Brant Myrold amp Sulzman 2006 Kramer et al 2010) These studiestheorized that root-derived carbon was the predominant source of carbon for microbesHowever this theory was not supported by a study in a mixed-wood forest of northernChina where microbes and soil enzyme activities were not significantly affected bythe interaction between nitrogen addition and plant inputs removal (Sun et al 2016)As resources for fungal decomposers nitrogen litter and roots were the main factorsinfluencing AMF and Sap Meanwhile bacteria mediated soil carbon processes via thecombined effects of nitrogen addition and plant inputs removal This was inconsistent withresults from M laosensis soils where root exclusion did not change the biomass of AMF(Wan et al 2015) Our results show that the biomass of GP was significantly influencedby nitrogen addition and plant inputs removal while the biomass of GN was significantlyaffected by plant inputs removal and not by nitrogen addition It is likely that GN firstutilizes recent plant material with a lower CN ratio as its carbon source while GP tendsto utilize more recalcitrant carbon first (Kramer amp Gleixner 2006)

Nitrogen addition drives linkage between soil microbial communitystructure and soil carbon in different plant inputs removalIt has been theorized that the availability of nitrogen can modulate the microbial structure-function relationship (Nguyen et al 2018) Here we found that nitrogen addition lead tothe direct and positive effect of GN on oxidases and hydrolases Meanwhile fungi (Sap)did not directly affect hydrolases This result was also observed in the literature wheresoil microbial community structure was not correlated with hydrolases under land-usechange conditions (Tischer Blagodatskaya amp Hamer 2015) GN had a strong total effecton soil oxidases and hydrolases under nitrogen addition while Sap had little effect Thisresult was inconsistent with previous studies that suggest that soil enzyme activities arenot always related to soil microbial community structure such as those in the area ofP-limitation (Tischer Blagodatskaya amp Hamer 2014) After plant inputs removal loweredthe carbon input the direct effects of bacteria and fungi varied under nitrogen additionHowever the GN in each plant inputs removal directly affected the oxidase activities It hasbeen theorized that substrate complexity leads to the connection between soil microbialcommunity and function (Baldrian 2009) We also found that the total effect of GNincreased PER activity and that the effect of Sap decreased PER activity in both the NL andNRL treatments It has been suggested that decreases in carbon input weaken the effectsof oxidases on complex carbon sources while the carbon decreases enhance the effects ofhydrolases on easily-decomposed carbon sources This idea has been supported by previousstudies showing that litter input increases lignin oxidase (Sun et al 2016) Meanwhile GNgrew quickly to compete for soil organic compounds after altering carbon and nitrogenresources This was consistent with previous studies (Bell et al 2009 Waldrop Balser amp

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Firestone 2000) that found that GN was correlated with hydrolases that degrade simpleorganic compounds

Presently understanding the linkages between soil enzyme activities and soil carbonproperties is necessary for predicting shifts in soil microbial community and functions(Weand et al 2010) Previous work has reported that soil enzyme activities are significantlycorrelated to SOC and MBC (Wang et al 2013) which was consistent with our result thatPER and BG activities directly and indirectly affected MBC and SOC under nitrogenaddition with and without plant inputs removal After the plant inputs removal the directeffect onMBC and SOC changed This was likely due to the functional shift from degradingcomplex to simple carbon resources These results show that soil oxidases greatly influencedsoil carbon properties under nitrogen addition However plant inputs removal enhancedthe total effect of BG on soil carbon properties which suggests that carbon acquisitionwas enhanced Our finding is consistent with previous studies theorizing that nitrogenaddition enhances linkages between oxidases and the loss of aliphatic carbon which arerelated to decreases in soil carbon storage (Cusack et al 2011) Meanwhile the interactionbetween nitrogen addition and plant inputs removal converted poorer-quality soil (iehigh lignin and low N) to higher-quality soil (ie low lignin and high N) which increasedmicrobial substrate utilization and strengthened the correlation between the hydrolasesand soil carbon properties (Ostertag et al 2008 Gallo et al 2005)

The regulation of soil carbon properties under treatments of nitrogenaddition with plant inputs removalResearch on the response of soil microbial community structure to ecosystem functionhas been growing rapidly (Lewis et al 2010 Malchair et al 2010) An increasing numberof studies have been published in recent years on the linkages between soil microbialcommunity structure functions and soil carbon dynamics (Cusack et al 2011 Ren et al2018 Sun et al 2017 Veres et al 2015) Variation in soil microbial communities is likelyto lead to microbial function shifts Specific soil microbial composition is involved inregulating specific soil enzyme activities which are used to assess the function of soilcarbon transformation and turnover (Waldrop amp Firestone 2004) Our results suggest thatshifts in soil microbial community structure mediated microbial functions and that theirinteractions regulated soil carbon turnover under treatments of nitrogen addition withplant inputs removal Our results are an important addition to the literature on this topic(You et al 2014 Brockett Prescott amp Grayston 2012)

In this paper the biomass of GN and the activity of PER were found to significantlyaffect soil carbon properties under nitrogen addition The activity of PER increased withthe biomass of GN at higher levels of nitrogen addition which enhanced the response ofsoil carbon to linkages between oxidases and bacteria (Cusack et al 2011) The changes incarbon inputs in the form of litter and root exudates altered soil microbial communityutilization and influenced functional shifts in soil enzymes (Baumann et al 2013 Tardy etal 2015)

Our results also suggest that the interactions between nitrogen addition and plantinputs removal significantly affected GN Furthermore the biomass of GN and Sap directly

Wu et al (2019) PeerJ DOI 107717peerj7343 1522

regulated soil enzyme activities and soil carbon properties because Sap was correlated withbelow- and above-ground carbon decomposition under plant inputs removal (Boldtburischet al 2018 Bennett et al 2017) GN and Sap regulated SOC by affecting on the activity ofPER which was involved in lignin degradation However some previous have reportedthat oxidases are produced directly by fungi (Jiang Cao amp Zhang 2014 Zhang et al 2014)

CONCLUSIONOur study revealed that moderate nitrogen addition increased soil microbial biomass Theinteraction between nitrogen addition and plant inputs removal significantly affected soilmicrobial community structure and function The biomass of gram-negative bacteria andsaprotrophic fungi directly affected how soil microbial function relates to carbon turnoverAltogether microbial community structure and function the biomass of gram-negativebacteria and peroxidase activity were the key factors regulating soil carbon dynamics Thisstudy suggests that nitrogen addition and plant inputs removal could alter soil enzymeactivities and further affect soil carbon turnover via microbial regulations These findingshave important implications for forest management The increase in soil microbial biomassand the microbial regulation on soil carbon both need to be considered when developingsustainable forest management practices for northern China Moreover further studies arealso needed to more precisely understand the complex interaction between the plant andthe below-ground processes and how it affects soil microbial community structure

ADDITIONAL INFORMATION AND DECLARATIONS

FundingThis study was financed by the National Key Research and Development Program of China(2016YFD0600205) The funders had no role in study design data collection and analysisdecision to publish or preparation of the manuscript

Grant DisclosuresThe following grant information was disclosed by the authorsNational Key Research and Development Program of China 2016YFD0600205

Competing InterestsThe authors declare there are no competing interests

Author Contributionsbull Ran Wu conceived and designed the experiments performed the experiments analyzedthe data prepared figures andor tables authored or reviewed drafts of the paperapproved the final draftbull Xiaoqin Cheng and Hairong Han conceived and designed the experiments contributedreagentsmaterialsanalysis tools authored or reviewed drafts of the paper approved thefinal draftbull Wensong Zhou performed the experiments approved the final draft

Wu et al (2019) PeerJ DOI 107717peerj7343 1622

Data AvailabilityThe following information was supplied regarding data availability

The raw measurements are available in the Supplemental Files

Supplemental InformationSupplemental information for this article can be found online at httpdxdoiorg107717peerj7343supplemental-information

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