Minor responses of soil microbial biomass, community structure and enzyme activities to nitrogen and phosphorus addition in three grassland ecosystems

  • Xiao Chen
  • Baihui Hao
  • Xin Jing
  • Jin-Sheng He
  • Wenhong Ma
  • Biao ZhuEmail author
Regular Article


Background and aims

Human activities have significantly increased nitrogen (N) and phosphorous (P) inputs to terrestrial ecosystems. However, the impact of N and P enrichment on soil microbial community structure and functioning in temperate and alpine grassland ecosystems remains unclear.


In this study, we investigated the responses of soil microbial communities to nutrient (N and P) additions in two temperate and one alpine grassland ecosystems in China. We measured soil chemical properties, microbial community composition (indicated by the phospholipid fatty acids, PLFA) and potential enzyme activities related to carbon (C), N, and P cycling in the peak growing season after 4 years of nutrient addition.


We found that N addition reduced soil pH and increased soil total N content at two meadow sites, P addition increased soil total P content at all three sites, but both N and P additions had minimal effects on soil organic C content. Bacteria and total microbial abundances did not change after N and P additions, while fungi and arbuscular mycorrhizal fungi (AMF) abundances were suppressed by N addition. Moreover, the activity of soil extracellular enzymes involved in C, N and P cycling and their stoichiometric ratios were not responsive to N and P additions, except for inhibition of acid phosphatase by P addition at the temperate meadow site.


Despite significant changes in soil chemistry (e.g., pH and available nutrients), soil microbial biomass (except fungi and AMF abundances), community structure, and enzyme activities (except phosphatase) were generally resistant to 4 years of N and P addition in the three temperate and alpine grassland ecosystems in China.


Grassland Phospholipid fatty acids Arbuscular mycorrhizal fungi Soil extracellular enzymes Nitrogen addition Phosphorus addition 



This study was supported by the National Natural Science Foundation of China (31622013, 31621091, 31630009, and 31370454). We thank Chao Wang, Wen Xiao and Huifeng Hu for their assistance in the field sampling and lab measurement. We also sincerely thank three anonymous reviewers and the Editor Dr. Sven Marhan for their helpful comments and suggestions that greatly improved the manuscript.

Supplementary material

11104_2019_4250_MOESM1_ESM.docx (992 kb)
ESM 1 (DOCX 991 kb)


  1. Allison SD, Vitousek PM (2005) Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biol Biochem 37:937–944CrossRefGoogle Scholar
  2. Bates D, Mächler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4. J Stat Softw 67(1):1–48CrossRefGoogle Scholar
  3. Bobbink R, Hicks K, Galloway J, Spranger T, Alkemade R, Ashmore M, Bustamante M, Cinderby S, Davidson E, Dentener F, Emmett B, Erisman J, Fenn M, Gilliam F, Nordin A, Pardo L, De Vries W (2010) Global assessment of nitrogen deposition effects on terrestrial plant diversity: a synthesis. Ecol Appl 20:30–59CrossRefPubMedGoogle Scholar
  4. Borer ET, Harpole WS, Adler PB, Lind EM, Orrock JL, Seabloom EW, Smith MD (2014) Finding generality in ecology: a model for globally distributed experiments. Methods Ecol Evol 5:65–73CrossRefGoogle Scholar
  5. Bossio DA, Scow KM (1998) Impacts of carbon and flooding on soil microbial communities: phospholipid fatty acid profiles and substrate utilization patterns. Microb Ecol 35:265–278CrossRefPubMedGoogle Scholar
  6. Burns RG, DeForest JL, Marxsen J, Sinsabaugh RL, Stromberger ME, Wallenstein MD, Weintraub MN, Zoppini A (2013) Soil enzymes in a changing environment: current knowledge and future directions. Soil Biol Biochem 58:216–234CrossRefGoogle Scholar
  7. Cenini VL, Fornara DA, McMullan G, Ternan N, Lajtha K, Crawley MJ (2015) Chronic nitrogen fertilization and carbon sequestration in grassland soils: evidence of a microbial enzyme link. Biogeochemistry 126:301–313CrossRefGoogle Scholar
  8. Chen D, Xing W, Lan Z, Saleem M, Wu Y, Hu S, Bai Y (2019) Direct and indirect effects of nitrogen enrichment on soil organisms and carbon and nitrogen mineralization in a semi-arid grassland. Funct Ecol 33:175–187CrossRefGoogle Scholar
  9. Chen DM, Lan ZC, Hu SJ, Bai YF (2015) Effects of nitrogen enrichment on belowground communities in grassland: Relative role of soil nitrogen availability vs. soil acidification. Soil Biol Biochem 89:99–108Google Scholar
  10. Chen X, Ding Z, Tang M, Zhu B (2018) Greater variations of rhizosphere effects within mycorrhizal group than between mycorrhizal group in a temperate forest. Soil Biol Biochem 126:237–246CrossRefGoogle Scholar
  11. Cleveland CC, Liptzin D (2007) C:N:P stoichiometry in soil: is there a “Redfield ratio” for the microbial biomass? Biogeochemistry 85:235–252CrossRefGoogle Scholar
  12. Crowther TW, Riggs C, Lin EM et al (2019) Sensitivity of global soil carbon stocks to combined nutrient enrichment. Ecol Lett 22:936–945CrossRefPubMedGoogle Scholar
  13. Cusack DF, Silver WL, Torn MS, Burton SD, Firestone MK (2011) Changes in microbial community characteristics and soil organic matter with nitrogen additions in two tropical forests. Ecology 92:621–632CrossRefPubMedGoogle Scholar
  14. Falkowski PG, Fenchel T, Delong EF (2008) The microbial engines that drive Earth's biogeochemical cycles. Science 320:1034–1039CrossRefPubMedGoogle Scholar
  15. Galloway JN, Dentener FJ, Capone DG, Boyer EW, Howarth RW, Seitzinger SP, Asner GP, Cleveland CC, Green PA, Holland EA, Karl DM, Michaels AF, Porter JH, Townsend AR, Vöosmarty CJJB (2004) Nitrogen cycles: past, present, and future. Biogeochemistry 70:153–226CrossRefGoogle Scholar
  16. German DP, Weintraub MN, Grandy AS, Lauber CL, Rinkes ZL, Allison SD (2011) Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biol Biochem 43:1387–1397CrossRefGoogle Scholar
  17. He D, Xiang X, He J-S, Wang C, Cao G, Adams J, Chu H (2016) Composition of soil fungal community is more sensitive to phosphorus than nitrogen addition in alpine meadow on the Qinghai-Tibetan plateau. Biol Fertil Soils 52:1059–1072CrossRefGoogle Scholar
  18. He NP, Yu Q, Wang RM, Zhang YH, Gao Y, Yu GR (2013) Enhancement of carbon sequestration in soil in the temperature grasslands of northern China by addition of nitrogen and phosphorus. PLoS One 8(10): e77241.
  19. He X, Ma W, Liang C, Hong M, Chai X, Zhao B, Zhang Y, Yang S, Zhang J, Xin X (2015) Effects of nutrient additions on community biomass varied among different grassland ecosystems of Inner Mongolia. Acta Sci Nat Univ Pekin 51:657–666 (in Chinese)Google Scholar
  20. Hungate BA, Jackson RB, Field CB, Chapin FS III (1996) Detecting changes in soil carbon in CO2 enrichment experiments. Plant Soil 187:135–145Google Scholar
  21. Jansa J, Mozafar A, Anken T, Ruh R, Sanders I, Frossard E (2002) Diversity and structure of AMF communities as affected by tillage in a temperate soil. Mycorrhiza 12:225–234CrossRefPubMedGoogle Scholar
  22. Jing X, Chen X, Tang M, Ding Z, Jiang L, Li P, Ma S, Tian D, Xu L, Zhu J, Ji C, Shen H, Zheng C, Fang J, Zhu B (2017) Nitrogen deposition has minor effect on soil extracellular enzyme activities in six Chinese forests. Sci Total Environ 607-608:806–815CrossRefPubMedGoogle Scholar
  23. Jing X, Chen X, Xiao W, Lin L, Wang C, He J-S, Zhu B (2018) Soil enzymatic responses to multiple environmental drivers in the Tibetan grasslands: insights from two manipulative field experiments and a meta-analysis. Pedobiologia 71:50–58CrossRefGoogle Scholar
  24. Jing X, Sanders NJ, Shi Y, Chu HY, Classen AT, Zhao K, Chen LT, Shi Y, Jiang YX, He JS (2015) The links between ecosystem multifunctionality and above- and belowground biodiversity are mediated by climate. Nat Commun 6:8159 | CrossRefPubMedPubMedCentralGoogle Scholar
  25. Keeler BL, Hobbie SE, Kellogg LE (2009) Effects of Long-term nitrogen addition on microbial enzyme activity in eight forested and grassland sites: implications for litter and soil organic matter decomposition. Ecosystems 12:1–15CrossRefGoogle Scholar
  26. Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microbiol 75:5111–5120CrossRefPubMedPubMedCentralGoogle Scholar
  27. Leff JW, Jones SE, Prober SM, Barberan A, Borer ET, Firn JL, Harpole WS, Hobbie SE, Hofmockel KS, Knops JM, McCulley RL, La Pierre K, Risch AC, Seabloom EW, Schutz M, Steenbock C, Stevens CJ, Fierer N (2015) Consistent responses of soil microbial communities to elevated nutrient inputs in grasslands across the globe. Proc Natl Acad Sci U S A 112:10967–10972CrossRefPubMedPubMedCentralGoogle Scholar
  28. Li Y, Liu Y, Wu S, Nie C, Lorenz N, Lee NR, Dick RP (2018) Composition and carbon utilization of soil microbial communities subjected to long-term nitrogen fertilization in a temperate grassland in northern China. Appl Soil Ecol 124:252–261CrossRefGoogle Scholar
  29. Ling N, Chen D, Guo H, Wei J, Bai Y, Shen Q, Hu S (2017) Differential responses of soil bacterial communities to long-term N and P inputs in a semi-arid steppe. Geoderma 292:25–33CrossRefGoogle Scholar
  30. Liu HM, Zhang AL, Huang CH, Li J, Wang H, Yang DL (2017) Effects of increasing nitrogen deposition on soil microbial community structure of Stipa Baicalensis steppe in Inner Mongolia, China. Ecol Environ Sci 26:1100–1106Google Scholar
  31. Liu LL, Greaver TL (2010) A global perspective on belowground carbon dynamics under nitrogen enrichment. Ecol Lett 13:819–828CrossRefPubMedGoogle Scholar
  32. Liu X, Zhang Y, Han W, Tang A, Shen J, Cui Z, Vitousek P, Erisman JW, Goulding K, Christie P, Fangmeier A, Zhang F (2013) Enhanced nitrogen deposition over China. Nature 494:459–462CrossRefPubMedGoogle Scholar
  33. Liu Y, Shi G, Mao L, Cheng G, Jiang S, Ma X, An L, Du G, Collins Johnson N, Feng H (2012) Direct and indirect influences of 8 yr of nitrogen and phosphorus fertilization on Glomeromycota in an alpine meadow ecosystem. New Phytol 194:523–535CrossRefPubMedGoogle Scholar
  34. Lu M, Zhou X, Luo Y, Yang Y, Fang C, Chen J, Li B (2011) Minor stimulation of soil carbon storage by nitrogen addition: a meta-analysis. Agric Ecosyst Environ 140:234–244CrossRefGoogle Scholar
  35. Luo R, Fan J, Wang W, Luo J, Kuzyakov Y, He JS, Chu H, Ding W (2019) Nitrogen and phosphorus enrichment accelerates soil organic carbon loss in alpine grassland on the Qinghai-Tibetan plateau. Sci Total Environ 650:303–312CrossRefPubMedGoogle Scholar
  36. Margalef O, Sardans J, Fernández-Martínez M, Molowny-Horas R, Janssens IA, Ciais P, Goll D, Richter A, Obersteiner M, Asensio D, Peñuelas J (2017) Global patterns of phosphatase activity in natural soils. Sci Rep. 7: 1337 |
  37. Marklein AR, Houlton BZ (2012) Nitrogen inputs accelerate phosphorus cycling rates across a wide variety of terrestrial ecosystems. New Phytol 193:696–704CrossRefPubMedGoogle Scholar
  38. Massey PA, Creamer RE, Whelan MJ, Ritz K (2016) Insensitivity of soil biological communities to phosphorus fertilization in intensively managed grassland systems. Grass Forage Sci 71:139–152CrossRefGoogle Scholar
  39. Nemergut DR, Townsend AR, Sattin SR, Freeman KR, Fierer N, Neff JC, Bowman WD, Schadt CW, Weintraub MN, Schmidt SK (2008) The effects of chronic nitrogen fertilization on alpine tundra soil microbial communities: implications for carbon and nitrogen cycling. Environ Microbiol 10:3093–3105CrossRefPubMedGoogle Scholar
  40. Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D Minchin P, O’Hara RB, Simpson G, Solymos P, Stevens MHH, Szoecs E, Wagner H (2019) Vegan: community ecology package. R package version 2.5–4. Accessed 20 Feb 2019
  41. Olander LP, Vitousek PM (2000) Regulation of soil phosphatase and chitinase activity by N and P availability. Biogeochemistry 49:175–190CrossRefGoogle Scholar
  42. Peñuelas J, Sardans J, Estiarte M, Ogaya R, Carnicer J, Coll M, Barbeta A, Rivas-Ubach A, Llusià J, Garbulsky M, Filella I, Jump AS (2013) Evidence of current impact of climate change on life: a walk from genes to the biosphere. Glob Chang Biol 19:2303–2338CrossRefPubMedGoogle Scholar
  43. R Development Core Team (2016) R: a language and environment for statistical computing. R Foundation for statistical computing, Vienna, Austria. ISBN 3-900051-07-0. Accessed 20 Feb 2019
  44. Revelle W (2018) psych: Procedures for personality and psychological research, Northwestern University, Evanston, Illinois, USA. R package version 18.12. Accessed 20 Feb 2019
  45. Riggs CE, Hobbie SE (2016) Mechanisms driving the soil organic matter decomposition response to nitrogen enrichment in grassland soils. Soil Biol Biochem 99:54–65CrossRefGoogle Scholar
  46. Riggs CE, Hobbie SE, Bach EM, Hofmockel KS, Kazanski CE (2015) Nitrogen addition changes grassland soil organic matter decomposition. Biogeochemistry 125:203–219CrossRefGoogle Scholar
  47. Rosseel Y (2012) Lavaan: An R package for structural equation modeling. J Stat Softw 48:1–36CrossRefGoogle Scholar
  48. Rousk J, Brookes PC, Baath E (2009) Contrasting soil pH effects on fungal and bacterial growth suggest functional redundancy in carbon mineralization. Appl Environ Microbiol 75:1589–1596CrossRefPubMedPubMedCentralGoogle Scholar
  49. Schimel DS (1995) Terrestrial ecosystems and the carbon cycle. Glob Chang Biol 1:77–91CrossRefGoogle Scholar
  50. Schimel JP, Weintraub MN (2003) The implications of exoenzyme activity on microbial carbon and nitrogen limitation in soil: a theoretical model. Soil Biol Biochem 35:549–563CrossRefGoogle Scholar
  51. Schrumpf M, Schulze ED, Kaiser K, Schumacher J (2011) How accurately can soil organic carbon stocks and stock changes be quantified by soil inventories? Biogeosciences 8:1193–1212CrossRefGoogle Scholar
  52. Sinsabaugh RL (2010) Phenol oxidase, peroxidase and organic matter dynamics of soil. Soil Biol Biochem 42:391–404CrossRefGoogle Scholar
  53. Sinsabaugh RL, Belnap J, Rudgers J, Kuske CR, Martinez N, Sandquist D (2015) Soil microbial responses to nitrogen addition in arid ecosystems. Front Microbiol 6:819–819CrossRefPubMedPubMedCentralGoogle Scholar
  54. Sinsabaugh RL, Lauber CL, Weintraub MN, Ahmed B, Allison SD, Crenshaw C, Contosta AR, Cusack D, Frey S, Gallo ME, Gartner TB, Hobbie SE, Holland K, Keeler BL, Powers JS, Stursova M, Takacs-Vesbach C, Waldrop MP, Wallenstein MD, Zak DR, Zeglin LH (2008) Stoichiometry of soil enzyme activity at global scale. Ecol Lett 11:1252–1264CrossRefPubMedGoogle Scholar
  55. Sinsabaugh RL, Moorhead DL (1994) Resource allocation to extracellular enzyme production: a model for nitrogen and phosphorus control of litter decomposition. Soil Biol Biochem 26:1305–1311CrossRefGoogle Scholar
  56. Tian D, Niu S (2015) A global analysis of soil acidification caused by nitrogen addition. Environ Res Lett 10:024019CrossRefGoogle Scholar
  57. Treseder KK (2008) Nitrogen additions and microbial biomass: a meta-analysis of ecosystem studies. Ecol Lett 11:1111–1120CrossRefPubMedGoogle Scholar
  58. van der Heijden MGA, Bardgett RD, van Straalen NM (2008) The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol Lett 11:296–310CrossRefPubMedGoogle Scholar
  59. Vitousek PM, Porder S, Houlton BZ, Chadwick OA (2010) Terrestrial phosphorus limitation: mechanisms, implications, and nitrogen-phosphorus interactions. Ecol Appl 20:5–15CrossRefPubMedGoogle Scholar
  60. Waldrop MP, Zak DR, Sinsabaugh RL, Gallo M, Lauber C (2004) Nitrogen deposition modifies soil carbon storage through changes in microbial enzymatic activity. Ecol Appl 14:1172–1177CrossRefGoogle Scholar
  61. Wang C (2018) The basic model of carbon balance in grassland ecosystem in northern China, and its response to nutrients addition and grazing, Ph.D. dissertation. Peking University, BeijingGoogle Scholar
  62. Wang P, Kong C, Sun B, Xu X (2010) Allantoin-induced changes of microbial diversity and community in rice soil. Plant Soil 332:357–368CrossRefGoogle Scholar
  63. Wieder WR, Cleveland CC, Smith WK, Todd-Brown K (2015) Future productivity and carbon storage limited by terrestrial nutrient availability. Nat Geosci 8:441–444CrossRefGoogle Scholar
  64. Xiao W, Chen X, Jing X, Zhu B (2018) A meta-analysis of soil extracellular enzyme activities in response to global change. Soil Biol Biochem 123:21–32CrossRefGoogle Scholar
  65. Yang S, Xu Z, Wang R, Zhang Y, Yao F, Zhang Y, Turco RF, Jiang Y, Zou H, Li H (2017) Variations in soil microbial community composition and enzymatic activities in response to increased N deposition and precipitation in inner Mongolian grassland. Appl Soil Ecol 119:275–285CrossRefGoogle Scholar
  66. Yang X, Ren F, Zhou H, He J (2014) Responses of plant community biomass to nitrogen and phosphorus additions in an alpine meadow on the Qinghai-Xizang plateau. Chinese Journal of Plant Ecology 38:159–166 (in Chinese)CrossRefGoogle Scholar
  67. Yuan X, Knelman JE, Gasarch E, Wang DL, Nemergut DR, Seastedt TR (2016) Plant community and soil chemistry responses to long-term nitrogen inputs drive changes in alpine bacterial communities. Ecology 97:1543–1554CrossRefPubMedGoogle Scholar
  68. Zechmeister-Boltenstern S, Keiblinger KM, Mooshammer M, Peñuelas J, Richter A, Sardans J, Wanek W (2015) The application of ecological stoichiometry to plant-microbial-soil organic matter transformations. Ecol Monogr 85:133–155CrossRefGoogle Scholar
  69. Zhang TA, Chen HYH, Ruan H (2018) Global negative effects of nitrogen deposition on soil microbes. ISME J 12:1817–1825CrossRefPubMedPubMedCentralGoogle Scholar
  70. Zhang YH, Han X, He NP, Long M, Huang JH, Zhang GM, Wang QB, Han XG (2014) Increase in ammonia volatilization from soil in response to N deposition in Inner Mongolia grasslands. Atmos Environ 84:156–162CrossRefGoogle Scholar
  71. Zhu J, Wang Q, He N, Smith MD, Elser JJ, Du J, Yuan G, Yu G, Yu Q (2016) Imbalanced atmospheric nitrogen and phosphorus depositions in China: implications for nutrient limitation. J Geophys Res Biogeosci 121:1605–1616CrossRefGoogle Scholar
  72. Zhou Z, Wang C, Zheng M, Jiang L, Luo Y (2017) Patterns and mechanisms of responses by soil microbial communities to nitrogen addition. Soil Biol Biochem 115:433–441CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Institute of Ecology, College of Urban and Environmental Sciences, and Key Laboratory for Earth Surface Processes of the Ministry of EducationPeking UniversityBeijingChina
  2. 2.School of Ecology and EnvironmentInner Mongolia UniversityHohhotChina
  3. 3.Rubenstein School of Environment and Natural ResourcesUniversity of VermontBurlingtonUSA
  4. 4.Gund Institute for EnvironmentUniversity of VermontBurlingtonUSA
  5. 5.State Key Laboratory of Grassland and Agro-ecosystems, College of Pastoral Agriculture Science and TechnologyLanzhou UniversityLanzhouChina

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