Biology and Fertility of Soils

, Volume 52, Issue 1, pp 101–112 | Cite as

Endospores, prokaryotes, and microbial indicators in arable soils from three long-term experiments

  • Paulina Tamez-Hidalgo
  • Bent T. Christensen
  • Mark A. Lever
  • Lars Elsgaard
  • Bente Aa. Lomstein
Original Paper


Management impacts on microbial communities in arable soil may influence soil quality and fertility. We examined the composition of the prokaryotic community in soils maintained under specific treatments for 24–118 years at Askov Experimental Station, Denmark. The experiments involved nutrient addition (unfertilized, mineral fertilizer, cattle manure), straw disposal (no straw, 8 t straw ha−1 year−1), and soil texture (5–18 % clay). Domain- and phylum-assigned cells were quantified by 16S ribosomal RNA (rRNA) gene analysis and endospores by analysis of dipicolinic acid (DPA). Amino sugars (glucosamine, galactosamine, and muramic acid) were assayed as microbial source indicators. Severe nutrient depletion reduced cell numbers and increased endospore abundance; straw disposal slightly increased both prokaryote and endospore numbers. Nutrient source (animal manure or mineral fertilizer) and soil texture had a little effect on cell and endospore numbers. With the notable exception of unfertilized soil, the ratio of endospores to total cells was similar across all treatments. The 16S rRNA gene analysis showed dominance of Bacteria over Archaea, the latter accounting for 0.2–8.4 % of total genes. Archaeal abundance differed a little among treatments. Firmicutes made up 0.2–1.2 % of the bacterial 16S rRNA genes. The numbers of Firmicutes were lower in unfertilized than in fertilized soil and decreased with decreasing soil clay content; straw treatment and nutrient source had a little effect. Amino sugar ratios suggested a dominance of fungi over bacteria, but the concentrations of microbial indicators and soil organic C were closely correlated, indicating that the amino sugar ratios represented a historical fingerprint (legacy effect) of the impact of management on the microbial community. Our results show that it takes extreme management to distort the general structure of prokaryotic communities in temperate arable soils.


Endospores Dipicolinic acid (DPA) Amino sugars Prokaryotes Fertilization Straw disposal Soil type 



This work was supported by the Mexican Research Council (CONACyT), PhD grant number 213154. The contribution of B.T.C. and L.E. and field experiments at Askov Experimental Station were financially supported by the EU-FP7 project SmartSOIL (Grant No. 289694). The research underlying this article has been cofunded by the Danish National Research Foundation and the European Research Council under the EU-FP7 (ERC Grant No. 294200). We acknowledge Lykke Poulsen, Susanne Nielsen, Jeanette Pedersen, Karina B. Henriksen, Trine B. Søgaard, and the staff at Askov Experimental Station for their excellent technical support.


  1. Acosta-Martínez V, Dowd S, Sun Y, Allen V (2008) Tag-encoded pyrosequencing analysis of bacterial diversity in a single soil type as affected by management and land use. Soil Biol Biochem 40:2762–2770CrossRefGoogle Scholar
  2. Amelung W, Brodowski S, Sandhage-Hofmann A, Bol R (2008) Combining biomarker with stable isotope analyses for assessing the transformation and turnover of soil organic matter. Adv Agron 100:155–250CrossRefGoogle Scholar
  3. Ammann AB, Kölle L, Brandl H (2011) Detection of bacterial endospores in soil by terbium fluorescence. Int J Microbiol 2011: Article ID 435281, 5 ppGoogle Scholar
  4. Bacchetti De Gregoris T, Aldred N, Clare AS, Burgess JG (2011) Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa. J Microbiol Methods 8:351–356CrossRefGoogle Scholar
  5. Bakken LR, Frostegard A (2006) Nucleic acid extraction from soil. In: Nannipieri P, Smalla K (Eds) Nucleic acids and proteins in soil. Soil biology, 8th edn. Springer, Berlin, pp 49–73Google Scholar
  6. Bengtson P, Sterngren AE, Rousk J (2012) Archaeal abundance across a pH gradient in an arable soil and its relationship to bacterial and fungal growth rates. Appl Environ Microbiol 78:5906–5911PubMedPubMedCentralCrossRefGoogle Scholar
  7. Bissett A, Richardson AE, Baker G, Thrall PH (2011) Long-term land use effects on soil microbial community structure and function. Appl Soil Ecol 51:66–78CrossRefGoogle Scholar
  8. Börjesson G, Menichetti L, Kirchmann H, Kätterer T (2012) Soil microbial community structure affected by 53 years of nitrogen fertilisation and different organic amendments. Biol Fertil Soils 48:245–257CrossRefGoogle Scholar
  9. Cadillo-Quiroz H, Bräuer S, Yashiro E, Sun C, Yavitt J, Zinder S (2006) Vertical profiles of methanogenesis and methanogens in two contrasting acidic peatlands in central New York State, USA. Environ Microbiol 8:1428–1440PubMedCrossRefGoogle Scholar
  10. Christensen BT, Bech-Andersen S (1989) Influence of straw disposal on distribution of amino acids in soil particle size fractions. Soil Biol Biochem 21:35–40CrossRefGoogle Scholar
  11. Christensen BT, Petersen J, Trentemøller U (2006) The Askov long-term experiments on animal manure and mineral fertilizers: the Lermarken site 1894–2004. DIAS Report Plant Production no. 121, April 2006, Danish Institute of Agricultural Sciences, Tjele, Denmark, pp. 104Google Scholar
  12. R Core Team (2014) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.
  13. Daims H, Bruhl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22:434–444PubMedCrossRefGoogle Scholar
  14. Enwall K, Nyberg K, Bertilsson S, Cederlund H, Stenström J, Hallin S (2007) Long-term impact of fertilization on activity and composition of bacterial communities and metabolic guilds in agricultural soil. Soil Biol Biochem 39:106–115CrossRefGoogle Scholar
  15. Fetzner S (1998) Bacterial degradation of pyridine, indole, quinolone, and their derivatives under different redox conditions. Appl Microbiol Biotechnol 49:237–350CrossRefGoogle Scholar
  16. Fichtel J, Köster J, Rullkötter J, Sass H (2007) Spore dipicolinic acid contents used for estimating the number of endospores in sediments. FEMS Microbiol Ecol 61:522–532PubMedCrossRefGoogle Scholar
  17. Fichtel J, Köster J, Rullkötter J, Sass H (2008) High variations in endospore numbers within tidal flat sediments revealed by quantification of dipicolinic acid. Geomicrobiol J 25:371–380CrossRefGoogle Scholar
  18. Filippidou S, Junier T, Wunderlin T, Lo C-C, Li P-E, Chain PS, Junier P (2015) Under-detection of endospore-forming Firmicutes in metagenomics data. Comp Struc Biotech J 13:299–306CrossRefGoogle Scholar
  19. Girvan MS, Bullimore J, Ball AS, Pretty JN, Osborn AM (2004) Responses of active bacterial and fungal communities in soils under winter wheat to different fertilizer and pesticide regimens. Appl Environ Microbiol 70:2692–2701PubMedPubMedCentralCrossRefGoogle Scholar
  20. Hartmann M, Fliessbach A, Oberholzer H-R, Widmer F (2006) Ranking the magnitude of crop and farming system effects on soil microbial biomass and genetic structure of bacterial communities. FEMS Microbiol Ecol 57:378–388PubMedCrossRefGoogle Scholar
  21. Janssen PH (2006) Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 72:1719–1728PubMedPubMedCentralCrossRefGoogle Scholar
  22. Joergensen RG, Wichern F (2008) Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biol Biochem 40:2977–2991CrossRefGoogle Scholar
  23. Johnson MJ, Lee KY, Scow KM (2003) DNA fingerprinting reveals links among agricultural crops, soil properties, and the composition of soil microbial communities. Geoderma 114:279–303CrossRefGoogle Scholar
  24. Kallmeyer J, Smith DC, Spivack AJ, D’Hondt S (2008) New cell extraction procedure applied to deep subsurface sediments. Limnol Oceanogr Meth 6:236–245CrossRefGoogle Scholar
  25. Karlsson AE, Johansson T, Bengtson P (2012) Archaeal abundance in relation to root and fungal exudation rates. FEMS Microbiol Ecol 80:305–311PubMedCrossRefGoogle Scholar
  26. Kristiansen SM, Hansen EM, Jensen LS, Christensen BT (2005) Natural 13C abundance and carbon storage in Danish soils under continuous silage maize. Eur J Agron 22:107–117CrossRefGoogle Scholar
  27. Langerhuus AT, Røy H, Lever MA, Morono Y, Inagaki F, Jørgensen BB, Lomstein BA (2012) Endospore abundance and D:L-amino acid modeling of bacterial turnover in Holocene marine sediment (Aarhus Bay). Geochim Cosmochim Acta 99:87–99CrossRefGoogle Scholar
  28. Lindroth P, Mopper K (1979) High performance liquid chromatographic determination of subpicomole amounts of amino-acids by precolumn fluorescence derivatization with o-phthaldialdehyde. Anal Chem 51:1667–1674CrossRefGoogle Scholar
  29. Lomstein BA, Jørgensen BB (2012) Pre-column liquid chromatographic determination of dipicolinic acid from bacterial endospores. Limnol Oceanogr Meth 10:227–233CrossRefGoogle Scholar
  30. Lomstein BA, Langerhuus AT, D’Hondt S, Jørgensen BB, Spivack AJ (2012) Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment. Nature 484:101–104PubMedCrossRefGoogle Scholar
  31. Loy A, Lehner A, Lee N, Adamczyk J, Meier H, Ernst J, Schleifer KH, Wagner M (2002) Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment. Appl Environ Microbiol 68:5064–5081PubMedPubMedCentralCrossRefGoogle Scholar
  32. Lunau M, Lemke A, Walther K, Martens-Habbena W, Simon M (2005) An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy. Environ Microbiol 7:961–968PubMedCrossRefGoogle Scholar
  33. Murugan R, Kumar S (2013) Influence of long-term fertilisation and crop rotation on changes in fungal and bacterial residues in a tropical rice-field soil. Biol Fertil Soils 49:847–856CrossRefGoogle Scholar
  34. Myrold DD, Nannipieri P (2014) Classical techniques versus omics approaches. In: Nannipieri P, Pietramellara G, Renella G (Eds) Omics in soil science. Caster Academy Press, Norfolk, pp 179–187Google Scholar
  35. Nannipieri P, Ascher J, Ceccherini MT, Landi L, Pietramellara G, Renella G (2003) Microbial diversity and soil functions. Eur J Soil Sci 54:655–670CrossRefGoogle Scholar
  36. Neumann D, Heuer A, Hemkemeyer M, Martens R, Tebbe CC (2013) Response of microbial communities to long-term fertilization depends on their microhabitat. FEMS Micobiol Ecol 86:71–84CrossRefGoogle Scholar
  37. Ogilvie LA, Hirsch PR, Johnston AWB (2008) Bacterial diversity of the Broadbalk Classical Winter Wheat Experiment in relation to long-term fertilizer inputs. Microb Ecol 56:525–537PubMedCrossRefGoogle Scholar
  38. Ollivier J, Töwe S, Bannert A, Hai B, Kastl E-M, Meyer A, Su MX, Kleineidam K, Schloter M (2011) Nitrogen turnover in soil and global change. FEMS Microbiol Ecol 78:3–16PubMedCrossRefGoogle Scholar
  39. Paterson E, Sim A, Osborne SM, Murray PJ (2011) Long-term exclusion of plant-inputs to soil reduces the functional capacity of microbial communities to mineralise root-derived carbon sources. Soil Biol Biochem 43:1873–1880CrossRefGoogle Scholar
  40. Paul EA (ed) (2015) Soil microbiology, ecology and biochemistry, 4th edn. Academic, LondonGoogle Scholar
  41. Pereira-e-Silva MC, Dias ACF, van Elsas JD, Salles JF (2012) Spatial and temporal variation of archaeal, bacterial and fungal communities in agricultural soils. PLoS One 7, e51554. doi: 10.1371/journal.pone.0051554 PubMedPubMedCentralCrossRefGoogle Scholar
  42. Portillo MC, Leff JW, Lauber CL, Fierer N (2013) Cell size distribution of soil bacteria and archaeal taxa. Appl Environ Microbiol 79:7610–7617PubMedPubMedCentralCrossRefGoogle Scholar
  43. Poulsen PHB, Al-Soud WA, Bergmark L, Magid J, Hansen LH, Sørensen SJ (2013) Effects of fertilization with urban and agricultural organic wastes in a field trial—prokaryotic diversity investigated by pyrosequencing. Soil Biol Biochem 57:784–793CrossRefGoogle Scholar
  44. Rodríguez-Blanco A, Sicardi M, Frioni L (2015) Plant genotype and nitrogen fertilization effects on abundance and diversity of diazotrophic bacteria associated with maize (Zea mays L.). Biol Fertil Soils 51:391–402CrossRefGoogle Scholar
  45. Rousk J, Bååth E, Brookes PC, Lauber CL, Lozupone C, Caporaso JG, Knight R, Fierer N (2010) Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME J 4:1340–1351PubMedCrossRefGoogle Scholar
  46. Schjønning P, Elmholt S, Christensen BT (eds) (2004) Managing soil quality—challenges in modern agriculture. CAB International, WallingfordGoogle Scholar
  47. Shen Z, Ruan Y, Chao X, Zhang J, Li R, Shen Q (2015) Rhizosphere microbial community manipulated by 2 years of consecutive biofertilizer application associated with banana Fusarium wilt disease suppression. Biol Fertil Soils 51:553–562CrossRefGoogle Scholar
  48. Stoddard SF, Smith BJ, Hein R, Roller BRK, Schmidt TM (2014) rrnDB: improved tools for interpreting rRNA gene abundance in bacteria and archaea and a new foundation for future development. Nuc Acid Res 43:D593–D598. doi: 10.1093/nar/gku1201 CrossRefGoogle Scholar
  49. Thomsen IK, Christensen BT (2004) Yields of wheat and soil carbon and nitrogen contents following long-term incorporation of barley straw and ryegrass catch crops. Soil Use Manag 20:432–438CrossRefGoogle Scholar
  50. Ulrich A, Becker R (2006) Soil parent material is a key determinant of the bacterial community structure in arable soils. FEMS Microbiol Ecol 56:430–443PubMedCrossRefGoogle Scholar
  51. Yin C, Jones KL, Peterson DE, Garrett KA, Hulbert SH, Paulitz TC (2010) Members of soil bacterial communities sensitive to tillage and crop rotation. Soil Biol Biochem 42:2111–2118CrossRefGoogle Scholar
  52. Yu Y, Lee C, Kim J, Hwang S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89:670–679PubMedCrossRefGoogle Scholar
  53. Zak DR, Holmes WE, White DC, Peacock AD, Tilman D (2003) Plant diversity, soil microbial communities, and ecosystem function: are there any links? Ecology 84:2042–2050CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Paulina Tamez-Hidalgo
    • 1
    • 5
  • Bent T. Christensen
    • 2
  • Mark A. Lever
    • 3
    • 4
  • Lars Elsgaard
    • 2
  • Bente Aa. Lomstein
    • 1
    • 3
  1. 1.Department of Bioscience, Section for MicrobiologyAarhus UniversityAarhus CDenmark
  2. 2.Department of AgroecologyAarhus UniversityTjeleDenmark
  3. 3.Department of Bioscience, Center for GeomicrobiologyAarhus UniversityAarhus CDenmark
  4. 4.Department of Environmental Systems Sciences, Institute for Biogeochemistry and Pollutant DynamicsETH ZurichZurichSwitzerland
  5. 5.Department for Animal Health and NutritionNovozymes A/SBagsvaerdDenmark

Personalised recommendations