Applied Microbiology and Biotechnology

, Volume 103, Issue 2, pp 995–1005 | Cite as

Diazotrophic microbial community and abundance in acidic subtropical natural and re-vegetated forest soils revealed by high-throughput sequencing of nifH gene

  • Han Meng
  • Zhichao Zhou
  • Ruonan Wu
  • Yongfeng Wang
  • Ji-Dong GuEmail author
Environmental biotechnology


Biological nitrogen fixation (BNF) is an important natural biochemical process converting the inert dinitrogen gas (N2) in the atmosphere to ammonia (NH3) in the N cycle. In this study, the nifH gene was chosen to detect the diazotrophic microorganisms with high-throughput sequencing from five acidic forest soils, including three natural forests and two re-vegetated forests. Soil samples were taken in two seasons (summer and winter) at two depth layers (surface and lower depths). A dataset of 179,600 reads obtained from 20 samples were analyzed to provide the microbial community structure, diversity, abundance, and relationship with physiochemical parameters. Both archaea and bacteria were detected in these samples and diazotrophic bacteria were the dominant members contributing to the biological dinitrogen fixation in the acidic forest soils. Cyanobacteria, Firmicutes, Proteobacteria, Spirocheates, and Verrucomicrobia were observed, especially the Proteobacteria as the most abundant phylum. The core genera were Bradyrhizobium and Methylobacterium from α-Proteobacteia, and Desulfovibrio from δ-Proteobacteia in the phylum of Proteobacteia of these samples. The diversity indices and the gene abundances of all samples were higher in the surface layer than the lower layer. Diversity was apparently higher in re-vegetated forests than the natural forests. Significant positive correlation to the organic matter and nitrogen-related parameters was observed, but there was no significant seasonal variation on the community structure and diversity in these samples between the summer and winter. The application of high-throughput sequencing method provides a better understanding and more comprehensive information of diazotrophs in acidic forest soils than conventional and PCR-based ones.


nifH gene High-throughput sequencing Nitrogen cycle Community Diversity Forest soil Southern China 



This study was funded by the National Natural Science Foundation of China (grant no. 31470562 to YFW), a Hong Kong PhD Fellowship (HM), and RGC GRF grant no. 701913 (J-DG).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_9466_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1199 kb)


  1. Andersen R, Chapman SJ, Artz RRE (2013) Microbial communities in natural and disturbed peatlands: a review. Soil Biol Biochem 57:979–994. CrossRefGoogle Scholar
  2. Bedmar E, Robles E, Delgado M (2005) The complete denitrification pathway of the symbiotic, nitrogen-fixing bacterium Bradyrhizobium japonicum. Biochem Soc Trans 33:141–144. CrossRefGoogle Scholar
  3. Bishop PE, Hawkins ME, Eady R (1986) Nitrogen fixation in molybdenum-deficient continuous culture by a strain of Azotobacter vinelandii carrying a deletion of the structural genes for nitrogenase (nifHDK). Biochem J 238:437–442. CrossRefGoogle Scholar
  4. Boyd ES, Hamilton TL, Peters JW (2011) An alternative path for the evolution of biological nitrogen fixation. Front Microbiol 2:205.
  5. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M (2012) Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6:1621–1624. CrossRefGoogle Scholar
  6. Chan OC, Casper P, Sha LQ, Feng ZL, Fu Y, Yang XD, Ulrich A, Zou XM (2008) Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis. FEMS Microbiol Ecol 64:449–458. CrossRefGoogle Scholar
  7. Chisnell JR, Premakumar R, Bishop PE (1988) Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J Bacteriol 170:27–33CrossRefGoogle Scholar
  8. Chistoserdova L, Chen SW, Lapidus A, Lidstrom ME (2003) Methylotrophy in Methylobacterium extorquens AM1 from a genomic point of view. J Bacteriol 185:2980–2987. CrossRefGoogle Scholar
  9. Chowdhury SP, Schmid M, Hartmann A, Tripathi AK (2009) Diversity of 16S-rRNA and nifH genes derived from rhizosphere soil and roots of an endemic drought tolerant grass, Lasiurus sindicus. Eur J Soil Biol 45:114–122. CrossRefGoogle Scholar
  10. Cleveland CC, Townsend AR, Schimel DS, Fisher H, Howarth RW, Hedin LO, Perakis SS, Latty EF, Von Fischer JC, Elseroad A, Wasson MF (1999) Global patterns of terrestrial biological nitrogen (N2) fixation in natural ecosystems. Glob Biogeochem Cycles 13:623–645. CrossRefGoogle Scholar
  11. Collavino MM, Tripp HJ, Frank IE, Vidoz ML, Calderoli PA, Donato M, Zehr JP, Aguilar OM (2014) nifH pyrosequencing reveals the potential for location-specific soil chemistry to influence N2-fixing community dynamics. Environ Microbiol 16:3211–3223. CrossRefGoogle Scholar
  12. Dos Santos PC, Fang Z, Mason SW, Setubal JC, Dixon R (2012). Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics 13:162.
  13. e Silva MCP, Semenov AV, van Elsas JD, Salles JF (2011) Seasonal variations in the diversity and abundance of diazotrophic communities across soils. FEMS Microbiol Ecol 77:57–68. CrossRefGoogle Scholar
  14. Fedorov DN, Doronina NV, Trotsenko YA (2011) Phytosymbiosis of aerobic methylobacteria: new facts and views. Microbiology 80:443–454. CrossRefGoogle Scholar
  15. Fierer N, Jackson RB (2006) The diversity and biogeography of soil bacterial communities. Proc Natl Acad Sci U S A 103:626–631. CrossRefGoogle Scholar
  16. Fish JA, Chai B, Wang Q, Sun Y, Brown CT, Tiedje JM, Cole JR (2013) FunGene: the functional gene pipeline and repository. Front Microbiol 4:291. CrossRefGoogle Scholar
  17. Flores-Mireles AL, Winans SC, Holguin G (2007) Molecular characterization of diazotrophic and denitrifying bacteria associated with mangrove roots. Appl Environ Microbiol 73:7308–7321. CrossRefGoogle Scholar
  18. Fujita Y, Takahashi Y, Chuganji M, Matsubara H (1992) The nifH-like (frxC) gene is involved in the biosynthesis of chlorophyll in the filamentous cyanobacterium Plectonema boryanum. Plant Cell Physiol 33:81–92Google Scholar
  19. Gaby JC, Buckley DH (2012) A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS One 7:e42149.
  20. 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 HJ, Vöosmarty CJ (2004) Nitrogen cycles: past, present, and future. Biogeochemistry 70:153–226. CrossRefGoogle Scholar
  21. Gan XH, Zhang FQ, Gu JD, Guo YD, Li ZQ, Zhang WQ, Xu XY, Zhou Y, Wen XY, Xie GG (2016) Differential distribution patterns of ammonia-oxidizing archaea and bacteria in acidic soils of Nanling National Nature Reserve forests in subtropical China. Antonie Van Leeuwenhoek 109:237–251. CrossRefGoogle Scholar
  22. Glass EM, Wilkening J, Wilke A, Antonopoulos D, Meyer F (2010) Using the metagenomics RAST server (MG-RAST) for analyzing shotgun metagenomes. Cold Spring Harb Protoc 2010(1):pdb. prot5368. CrossRefGoogle Scholar
  23. Graham PH (1992) Stress tolerance in Rhizobium and Bradyrhizobium, and nodulation under adverse soil conditions. Can J Microbiol 38:475–484. CrossRefGoogle Scholar
  24. Griffiths RI, Thomson BC, James P, Bell T, Bailey M, Whiteley AS (2011) The bacterial biogeography of British soils. Environ Microbiol 13:1642–1654. CrossRefGoogle Scholar
  25. Gully D, Sadowsky MJ, Giraud E, Xu L, Chaintreuil C, Gargani D (2013) Photosynthetic Bradyrhizobium sp. strain ORS285 is capable of forming nitrogen-fixing root nodules on soybeans (Glycine max). Appl Environ Microbiol 79:2459–2462. CrossRefGoogle Scholar
  26. Heidelberg JF, Seshadri R, Haveman SA, Hemme CL, Paulsen IT, Kolonay JF, Eisen JA, Ward N, Methe B, Brinkac LM (2004) The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Nature Biotechnol 22:554–559. CrossRefGoogle Scholar
  27. Homer MJ, Dean DR, Roberts GP (1995) Characterization of the γ protein and its involvement in the metallocluster assembly and maturation of dinitrogenase from Azotobacter vinelandii. J Biol Chem 270:24745–24752. CrossRefGoogle Scholar
  28. Jing HM, Xia XM, Liu HB, Zhou Z, Wu C, Nagarajan S (2015) Anthropogenic impact on diazotrophic diversity in the mangrove rhizosphere revealed by nifH pyrosequencing. Front Microbiol 6:1172. CrossRefGoogle Scholar
  29. Koponen P, Nygren P, Domenach AM, Le Roux C, Saur E, Roggy JC (2003) Nodulation and dinitrogen fixation of legume trees in a tropical freshwater swamp forest in French Guiana. J Trop Ecol 19:655–666. CrossRefGoogle Scholar
  30. 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 75:5111–5120. CrossRefGoogle Scholar
  31. Letunic I, Bork P (2007) Interactive tree of life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23:127–128. CrossRefGoogle Scholar
  32. Li H, Ye D, Wang X, Settles ML, Wang J, Hao Z, Zhou L, Dong P, Jiang Y, Ma ZS (2014) Soil bacterial communities of different natural forest types in Northeast China. Plant Soil 383:203–216. CrossRefGoogle Scholar
  33. Liao X, Inglett PW (2014) Dynamics of periphyton nitrogen fixation in short-hydroperiod wetlands revealed by high-resolution seasonal sampling. Hydrobiologia 722:263–277. CrossRefGoogle Scholar
  34. Lincoln NK, Vitousek P (2016) Nitrogen fixation during decomposition of sugarcane (Saccharum officinarum) is an important contribution to nutrient supply in traditional dryland agricultural systems of Hawai'i. Int J Agric Sustain 14:214–230. CrossRefGoogle Scholar
  35. Lu RK (2000) Methods of agriculture chemical analysis. China Agriculture Scientech Press, BeijingGoogle Scholar
  36. Magoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963. CrossRefGoogle Scholar
  37. McDermoti TR, Graham PH (1990) Competitive ability and efficiency in nodule formation of strains of Bradyrhizobium japonicum. Appl Environ Microbiol 56:3035–3039Google Scholar
  38. Mehta MP, Baross JA (2006) Nitrogen fixation at 92 °C by a hydrothermal vent archaeon. Science 314:1783–1786. CrossRefGoogle Scholar
  39. Meng H, Wang YF, Chan HW, Wu RN, Gu J-D (2016) Co-occurrence of nitrite-dependent anaerobic ammonium and methane oxidation processes in subtropical acidic forest soils. Appl Microbiol Biotechnol 5:1–13. Google Scholar
  40. Meng H, Wu RN, Wang YF, Gu J-D (2017) A comparison of denitrifying bacterial community structures and abundance in acidic soils between natural forest and re-vegetated forest of Nanling nature reserve in southern China. J Environ Manag 198:41–49. CrossRefGoogle Scholar
  41. Mergel A, Schmitz O, Mallmann T, Bothe H (2001) Relative abundance of denitrifying and dinitrogen-fixing bacteria in layers of a forest soil. FEMS Microbiol Ecol 36:33–42. CrossRefGoogle Scholar
  42. Mizuno M, Yurimoto H, Yoshida N, Iguchi H, Sakai Y (2012) Distribution of pink-pigmented facultative methylotrophs on leaves of vegetables. Biosci Biotechnol Biochem 76:578–580. CrossRefGoogle Scholar
  43. Moisander PH, Shiue L, Steward GF, Jenkins BD, Bebout BM, Zehr JP (2006) Application of a nifH oligonucleotide microarray for profiling diversity of N2-fixing microorganisms in marine microbial mats. Environ Microbiol 8:1721–1735. CrossRefGoogle Scholar
  44. Moseman-Valtierra S, Levin LA, Martin RM (2016) Anthropogenic impacts on nitrogen fixation rates between restored and natural Mediterranean salt marshes. Mar Ecol 37:370–379. CrossRefGoogle Scholar
  45. Nomata J, Mizoguchi T, Tamiaki H, Fujita Y (2006) A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis reconstitution of chlorophyllide a reductase with purified x-protein (bchX) and yz-protein (bchY-bchZ) from Rhodobacter capsulatus. J Biol Chem 281:15021–15028. CrossRefGoogle Scholar
  46. Nüsslein K, Tiedje JM (1999) Soil bacterial community shift correlated with change from forest to pasture vegetation in a tropical soil. Appl Environ Microbiol 65:3622–3626Google Scholar
  47. Ormeño-Orrillo E, Rogel-Hernández MA, Lloret L, López-López A, Martínez J, Barois I, Martínez-Romero E (2012) Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico). Microb Ecol 63:822–834. CrossRefGoogle Scholar
  48. Poly F, Ranjard L, Nazaret S, Gourbière F, Monrozier LJ (2001) Comparison of nifH gene pools in soils and soil microenvironments with contrasting properties. Appl Environ Microbiol 67:2255–2262. CrossRefGoogle Scholar
  49. Radajewski S, Webster G, Reay DS, Morris SA, Ineson P, Nedwell DB, Prosser JI, Murrell JC (2002) Identification of active methylotroph populations in an acidic forest soil by stable-isotope probing. Microbiology 148:2331–2342. CrossRefGoogle Scholar
  50. Raymond J, Siefert JL, Staples CR, Blankenship RE (2004) The natural history of nitrogen fixation. Mol Biol Evol 21:541–554. CrossRefGoogle Scholar
  51. Sánchez C, Tortosa G, Granados A, Delgado A, Bedmar EJ, Delgado MJ (2011) Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding. Soil Biol Biochem 43:212–217. CrossRefGoogle Scholar
  52. Souillard N, Magot M, Possot O, Sibold L (1988) Nucleotide sequence of regions homologous to nifH (nitrogenase Fe protein) from the nitrogen-fixing archaebacteria Methanococcus thermolithotrophicus and Methanobacterium ivanovii: evolutionary implications. Mol Evol J 27:65–76. CrossRefGoogle Scholar
  53. Sparks DL, Page AL, Helmke PA, Loeppert RH, Soltanpour PN, Tabatabai MA, Johnston CT, Sumner ME (1996). Methods of soil analysis. part 3 - chemical methods. Wisconsin, USA.Google Scholar
  54. Stacheter A, Noll M, Lee CK, Selzer M, Glowik B, Ebertsch L, Mertel R, Schulz D, Lampert N, Drake HL (2013) Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs. ISME J 7:1051–1064. CrossRefGoogle Scholar
  55. Staples CR, Lahiri S, Raymond J, Von Herbulis L, Mukhophadhyay B, Blankenship RE (2007) Expression and association of group IV nitrogenase nifD and nifH homologs in the non-nitrogen-fixing archaeon Methanocaldococcus jannaschii. Bacteriol J 189:7392–7398CrossRefGoogle Scholar
  56. Steppe T, Paerl HW (2005) Nitrogenase activity and nifH expression in a marine intertidal microbial mat. Microb Ecol 49:315–324. CrossRefGoogle Scholar
  57. Turk-Kubo KA, Karamchandani M, Capone DG, Zehr JP (2014) The paradox of marine heterotrophic nitrogen fixation: abundances of heterotrophic diazotrophs do not account for nitrogen fixation rates in the eastern tropical South Pacific. Environ Microbiol 16:3095–3114. CrossRefGoogle Scholar
  58. US Department of Agriculture (1999) Soil taxonomy—a basic system of soil classification for making and interpreting soil surveys. USDA, Washington.Google Scholar
  59. Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA, Schindler DW, Schlesinger WH, Tilman DG (1997) Human alteration of the global nitrogen cycle: sources and consequences. Ecol Appl 7:737–750.[0737:HAOTGN]2.0.CO;2Google Scholar
  60. Wang Q, Quensen JF, Fish JA, Lee TK, Sun Y, Tiedje JM, Cole JR (2013) Ecological patterns of nifH genes in four terrestrial climatic zones explored with targeted metagenomics using FrameBot, a new informatics tool. MBio 4:e00592–e00513. Google Scholar
  61. Yamada A, Inoue T, Noda S, Hongoh Y, Ohkuma M (2007) Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites. Mol Ecol 16:3768–3777. CrossRefGoogle Scholar
  62. Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of EnvironmentNanjing Normal UniversityNanjingChina
  2. 2.Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, Faculty of ScienceThe University of Hong KongHong KongPeople’s Republic of China
  3. 3.Guangdong Provincial Key Laboratory of Silviculture, Protection and UtilizationGuangdong Academy of ForestryGuangzhouPeople’s Republic of China

Personalised recommendations