Bacterial Diversity in Livestock Manure Composts as Characterized by Terminal Restriction Fragment Length Polymorphisms (T-RFLP) of PCR-amplified 16s rRNA Gene Sequences

  • S. M. Tiquia
  • F. C. MichelJr.
Conference paper


Composts contain a large and diverse community of microorganisms that play a central role in the decomposition of organic matter during the composting process. However, microbial communities active in composts have not been well described in the past. In the present study, the phylogenetic diversity of bacterial communities in livestock manure compost was determined based on terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes. This technique uses a PCR in which one of the primers is fluorescently labeled. After amplification, the PCR product is then digested with restriction enzymes such as HhaI, MspI, and RsaI to generate T-RFLP fingerprints of bacterial communities. In the present study, a mixture of dairy and horse manure (dairy+horse manure; 1:1 ratio w/w) was composted in windrows and in-vessel to investigate compost bacterial diversity. The DNA was isolated from the feedstocks (day 0) and after 21 and 104 days of in-vessel and windrow composting, respectively, for T-RFLP analysis. A variety of techniques were then used to analyze T-RFLP data to gain insights about the structure of the bacterial community from these compost samples. Results of the T-RFLP analysis revealed high species diversity in the feedstocks sample As many as 27 to 39 different terminal restriction fragments (T-RFs) were found in these samples, revealing high diversity in the livestock manure composts. After composting, an increase in the T-RFLP-based Shannon diversity index was observed in the in-vessel compost, while a decrease was found in the windrow compost. Differences in chemical properties were also observed in the windrow and in-vessel composts. The windrow compost had lower water, organic matter (OM) and C contents and higher C and OM loss than the in-vessel compost.


Bacterial Community Terminal Restriction Fragment Length Polymorphism Shannon Diversity Index Livestock Manure Compost Sample 
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  1. Atlas RM, Bartha R (1997) Microbial ecology: fundamentals and applications, 4th edn. Benjamin/Cummings, Menlo Park, California, 694 ppGoogle Scholar
  2. Beffa T, Blanc M, Marilley L, Fischer JL, Lyon,PF, Aragno M (1996) Taxonomic and metabolic microbial diversity during composting. In: De Bertoldi M, Sequi P, Lemmes B, Papi T (eds) The science of composting. Part I. M. Chapman and Hall, London, pp 149 - 161Google Scholar
  3. Blanc M, Beffa T, Aragno M (1996) Biodiversity of thermophilic bacteria isolated from hot compost piles. In: De Bertoldi M, Sequi P, Lemmes B, Papi T (eds) The science of composting. Part II. Chapman and Hall, London, pp 1087 - 1090Google Scholar
  4. Clement BG, Kehl LE, DeBord KL, Kits CL (1998) Terminal restriction fragment patterns ( TRFLPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J Microbiol Methods 31: 135-142Google Scholar
  5. Epstein E (1997) The Science of Composting. Technomic Publishing, Lancaster, Pennsylvania, 487 ppGoogle Scholar
  6. Fujio Y, Kume SJ (1991) Isolation and identification of thermophilic bacteria from sewage sludge compost. J Ferment Bioeng 72: 334 - 337CrossRefGoogle Scholar
  7. Golueke CG (1972) Composting: a study of the process and its principles. Rodale Press, Emmaus, Pennsylvania, 110 ppGoogle Scholar
  8. Hammer PL, Rudeanu S (1968) Boolean methods in operation research and related areas. Spinger, New York 329 ppCrossRefGoogle Scholar
  9. Heuer H, Smalla K (1997) Application of denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis for studying soil microbial communities. In: Van Elsas JD, Trevors JT, Wellington EMH (eds) Modern Soil Microbiology. Marcel Dekker, New York, pp 352 - 373Google Scholar
  10. Keener HM, Elwell DL, Reid GL, Michel FC Jr (2000) Composting non-separated dairy manure-theoretical limits and practical experience. Proc 8th Int Symp on Animal, Agricultural and Food Processing Wastes (ISAAFPW 2000 ). Des Moines, IowaGoogle Scholar
  11. Kerkhof L, Santoro M, Garland J (2000) Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism ( TRFLP) analysis. FEMS Microbiol Lett 184: 95-101Google Scholar
  12. Liu WT, Marsh T, Cheng H, Forney LJ (1997) Characterization of microbial community by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl Environ Microbiol 63: 4516 - 4522PubMedGoogle Scholar
  13. Ludemann H, Arth I, Liesack W (2000) Spatial changes in bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores. Appl Environ Microbiol 66: 754 - 762PubMedCrossRefGoogle Scholar
  14. Maidak BL (2000) The Ribosomal Database Project II. Abstract presented at the Midwest Molecular Ecology 2000 Conference. July 16-18, 2000, Northern Illinois University, Illinois, p 16Google Scholar
  15. Marsh TL, Saxman P, Cole J, Tiedje J (2000) Terminal restriction fragment length polymorphism analysis program, a web-based research tool for microbial community analysis. Appl Environ Microbiol 66: 3616 - 3620PubMedCrossRefGoogle Scholar
  16. Massol-Deya AA, Odelson DA, Hickey RF, Tiedjie JM (1995) Bacterial community fingerprinting of amplified 16S and 16-23S ribosomal gene sequences and restriction endonucleases analysis (ARDRA). Mol Microb Ecol Man 3: 3: 2: 1 - 8Google Scholar
  17. Pielou EC (1969) An introduction to mathematical ecology. Wiley, New YorkGoogle Scholar
  18. Schwieger F, Tebbe CC (1998) A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene based microbial community analysis. Appl Environ Microbiol 64: 4870 - 4876PubMedGoogle Scholar
  19. Shannon CE, Weaver W (1949) The mathematical theory of communication. University of Illinois Press, Urbana, Illinois, 125 ppGoogle Scholar
  20. Sparks DL (1996) Nitrogen-total. In: Methods of soil analysis. Part 3- Chemical methods. SSSA, Madison, WisconsinGoogle Scholar
  21. Strom PF (1985a) Identification of thermophilic bacteria in solid waste composting. Appl Environ Microbiol 50: 906 - 913PubMedGoogle Scholar
  22. Strom PF (1985b) Effect of temperature on bacterial species diversity in thermophilic solid-waste composting. Appl Environ Microbiol 50: 899 - 905PubMedGoogle Scholar
  23. Suzuki MT, Giovanni SJ (1996) Bias caused by template annealing in the amplication of mixtures of 16S rRNA genes by PCR. Appl Envrion Microbiol 62: 625 - 630Google Scholar
  24. Tiquia SM, Tam NFY, Hodgkiss IJ (1996) Microbial activities during composting of spent pig-manure sawdust litter at different moisture contents. Biores Technol 55: 201 - 206CrossRefGoogle Scholar
  25. Wiener N (1948) Cybernetics or control and communication in the animal and the machine. The Massachusetts Institute of Technology Press, Cambridge, Massachusetts, 191 ppGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2002

Authors and Affiliations

  • S. M. Tiquia
    • 1
  • F. C. MichelJr.
    • 2
  1. 1.Environmental Sciences DivisionOak Ridge National LaboratoryOak RidgeUSA
  2. 2.Department of Food, Agricultural, and Biological EngineeringThe Ohio State University, Ohio Agricultural Research and Development Center (OARDC)WoosterUSA

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