Applied Microbiology and Biotechnology

, Volume 99, Issue 10, pp 4471–4484 | Cite as

Microbial diversity observed during hemp retting

  • Alexandra Ribeiro
  • Philippe Pochart
  • Arnaud Day
  • Sarah Mennuni
  • Pierre Bono
  • Jean-Luc Baret
  • Jean-Louis Spadoni
  • Irène Mangin
Environmental biotechnology


Historically used in textile and paper industry, hemp fibres have started to find new applications in composite materials with important economic and ecological advantages. However, their applications are limited since manufacturers have some difficulties to standardise fabrication processes. This study is a first step before selection and isolation of strains that could later be used to optimise microbial retting efficiency and hence fibre quality. We studied six samples harvested on different ground types, at different dates and with different retting durations on field to obtain an exhaustive representation of the process. After DNA extraction, total bacteria and fungi associated with stems during retting were specifically quantified using real-time PCR. Then, using sequence analysis of randomly cloned 16S and 18S ribosomal RNA (rRNA) genes, a phylogenetic characterisation of the dominant microorganisms was carried out. Quantitatively, we showed that there were 8.1–9.5 log10 16S rRNA gene copies per gram of hemp straw for bacteria and 8.6–9.6 log10 18S rRNA gene copies per gram for fungi. Qualitatively, we noticed a higher bacterial diversity in comparison to fungi. This work showed that in the different samples, the same species were present but in significantly different proportions according to ground type, harvest dates and retting durations on field. The most frequent bacterial sequences were affiliated to species Escherichia coli, Pantoea agglomerans, Pseudomonas rhizosphaerae, Rhodobacter sp., Pseudomonas fulva, Rhizobium huautlense and Massilia timonae, whereas fungal sequences were principally related to the genera Cladosporium and Cryptococcus.


Hemp retting Microbiota Molecular inventory qPCR 



We thank Ester Pereira for technical assistance in isolating bacterial and fungal strains for the future studies. We thank Antonia Suau for revising the manuscript. This work was financially supported by Fibres Recherche Développement®, France.

Conflict of interest

There is no conflict of interest.

Supplementary material

253_2014_6356_MOESM1_ESM.pdf (136 kb)
ESM 1 (PDF 135 kb)


  1. Acinas SG, Marcelino LA, Klepac-Ceraj V, Polz MF (2004) Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J Bacteriol 186:2629–2635CrossRefPubMedCentralPubMedGoogle Scholar
  2. Akin DE (2013) Linen most useful: perspective on structure, chemistry and enzyme for the retting flax. ISRN Biotechnology 2013:1–23CrossRefGoogle Scholar
  3. Akin DE, Rigsby LL, Henriksson G, Eriksson KL (1998) Structural effects on flax stems of three potential retting fungi. Text Res J 68:515–519CrossRefGoogle Scholar
  4. Alkorta I, Garbisu G, Llama MJ, Serra JL (1998) Industrial applications of pectic enzymes: a review. Process Biochem 33:21–28CrossRefGoogle Scholar
  5. Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedCentralPubMedGoogle Scholar
  6. Betrabet SM, Bhat JV (1958) Pectin decomposition by species of Pseudomonas and their role in the retting of malvaceous plants. Appl Microbiol 6:89–93PubMedCentralPubMedGoogle Scholar
  7. Borneman J, Hartin RJ (2000) PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol 66:4356–4360CrossRefPubMedCentralPubMedGoogle Scholar
  8. Bourmaud A (2013) Observation of the structure of a composite polypropylene/flax and damage mechanisms under stress. Ind Crop Prod 43:225–236CrossRefGoogle Scholar
  9. Cronier D, Monties B, Chabbert B (2005) Structure and chemical composition of bast fibers isolated from developing hemp stem. J Agric Food Chem 53:8279–8289CrossRefPubMedGoogle Scholar
  10. Das B, Chakrabarti K, Ghosh S, Chakraborty A, Saha MN (2013) Assessment of changes in community level physiological profile and molecular diversity of bacterial communities in different stages of jute retting. Pak J Biol Sci 16:1722–1729CrossRefPubMedGoogle Scholar
  11. Das B, Chakrabarti K, Ghosh S, Majumdar B, Tripathi S, Chakraborty A (2012) Effect of efficient pectinolytic bacterial isolates on retting and fibre quality of jute. Ind Crop Prod 36:415–417CrossRefGoogle Scholar
  12. Di Candilo M, Bonatti PM, Guidetti C, Focher B, Grippo C, Tamburini E, Mastromei G (2010) Effects of selected pectinolytic bacterial strains on water-retting of hemp and fibre properties. J Appl Microbiol 108:194–203CrossRefPubMedGoogle Scholar
  13. Fogarty M, Ward OP (1972) Pectic substances and pectolytic enzymes. Process Biochem 7:17–31Google Scholar
  14. Godon JJ, Zumstein E, Dabert P, Habouzit F, Moletta R (1997) Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 63:2802–2813PubMedCentralPubMedGoogle Scholar
  15. Good IJ (1953) The population frequencies of species and the estimation of population parameters. Biom J 40:237–264Google Scholar
  16. Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221–224CrossRefPubMedGoogle Scholar
  17. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321CrossRefPubMedGoogle Scholar
  18. Hayashi K, Inoue Y, Shiga M, Sato S, Takano R, Hirayae K, Hibi T, Hara S (1997) Pectinolytic enzymes from Pseudomonas marginalis MAFF 03-01173. Phytochemistry 45:1359–1363CrossRefPubMedGoogle Scholar
  19. Henriksson G, Akin DE, Hanlin RT, Rodriguez C, Archibald DD, Rigsby LL, Eriksson KL (1997) Identification and retting efficiencies of fungi isolated from dew-retted flax in the United States and Europe. Appl Environ Microbiol 63:3950–3956PubMedCentralPubMedGoogle Scholar
  20. Himmelsbach DS, Khalili S, Akin DE (2002) The use of FT-IR microspectroscopic mapping to study the effects of enzymatic retting of flax (Linum usitatissimum L) stems. J Sci Food Agric 82:685–696CrossRefGoogle Scholar
  21. Hoondal GS, Tiwari RP, Tewari R, Dahiya N, Beg QK (2002) Microbial alkaline pectinases and their industrial applications: a review. Appl Microbiol Biotechnol 59:409–418CrossRefPubMedGoogle Scholar
  22. Howlett BJ, Rolls BD, Cozijnsen AJ (1997) Organisation of ribosomal DNA in the ascomycete Leptosphaeria maculans. Microbiol Res 152:261–267CrossRefPubMedGoogle Scholar
  23. Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180:4765–4774PubMedCentralPubMedGoogle Scholar
  24. Klappenbach JA, Dunbar JM, Schmidt TM (2000) rRNA operon copy number reflects ecological strategies of bacteria. Appl Environ Microbiol 66:1328–1333CrossRefPubMedCentralPubMedGoogle Scholar
  25. Magne F, Abely M, Boyer F, Morville P, Pochart P, Suau A (2006a) Low species diversity and high interindividual variability in faeces of preterm infants as revealed by sequences of 16S rRNA genes and PCR-temporal temperature gradient gel electrophoresis profiles. FEMS Microbiol Ecol 57:128–138CrossRefPubMedGoogle Scholar
  26. Magne F, Hachelaf W, Suau A, Boudraa G, Mangin I, Touhami M, Bouziane-Nedjadi K, Pochart P (2006b) A longitudinal study of infant faecal microbiota during weaning. FEMS Microbiol Ecol 58:563–571CrossRefPubMedGoogle Scholar
  27. Maidak BL, Cole JR, Parker CT Jr, Garrity GM, Larsen N, Li B, Lilburn TG, McCaughey MJ, Olsen GJ, Overbeek R, Pramanik S, Schmidt TM, Tiedje JM, Woese CR (1999) A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res 27:171–173CrossRefPubMedCentralPubMedGoogle Scholar
  28. Marrot L, Lefeuvre A, Pontoire B, Bourmaud A, Baley C (2013) Analysis of the hemp fiber mechanical properties and their scattering (Fedora 17). Ind Crop Prod 51:317–327CrossRefGoogle Scholar
  29. Martin N, Mouret N, Davies P, Baley C (2013) Influence of the degree of retting of flax fibers on the tensile properties of single fibers and short fiber/polypropylene composites. Ind Crop Prod 49:755–767CrossRefGoogle Scholar
  30. Mazzaferro L, Pinuel L, Minig M, Breccia JD (2010) Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid beta-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria. Arch Microbiol 192:383–393CrossRefPubMedGoogle Scholar
  31. McGovern JN (1983) Fibres (vegetable). In: Grayson M (ed) Encyclopedia of composite materials and components. Wiley-Interscience, New York, pp 497–512Google Scholar
  32. Munshi TK, Chattoo BB (2008) Bacterial population structure of the jute-retting environment. Microb Ecol 56:270–282CrossRefPubMedGoogle Scholar
  33. Müssig J, Cescutti G, Fischer H (2006) Le management de la qualité appliqué à l’emploi des fibres naturelles dans l’industrie. In: Bouloc P (ed) Le chanvre industriel: production et utilisations. Editions France Agricole, Paris, pp 235–269 [in French]Google Scholar
  34. Naidu GSN, Panda T (1998) Production of pectolytic enzymes: a review. Bioprocess Eng 19:355–361CrossRefGoogle Scholar
  35. Pickering KL, Li Y, Farrel RL (2007) Fungal and alkali interfacial modification of hemp fibre reinforced composites. Key Eng Mater 334–335:493–496CrossRefGoogle Scholar
  36. Renault D, Vallance J, Deniel F, Wery N, Godon JJ, Barbier G, Rey P (2012) Diversity of bacterial communities that colonize the filter units used for controlling plant pathogens in soilless cultures. Microb Ecol 63:170–187CrossRefPubMedGoogle Scholar
  37. Rosemberg JA (1965) Bacteria responsible for the retting of Brazilian flax. Appl Microbiol 13:991–992PubMedCentralPubMedGoogle Scholar
  38. Sharma HSS, Robinson E (1983) Fungal colonization during glyphosate induced desiccation and dew-retting of flax cultivars. Technical report n 228111Google Scholar
  39. Sharma HSS (1986a) An alternative method of flax retting during dry weather. Ann Appl Biol 109:605–611CrossRefGoogle Scholar
  40. Sharma HSS (1986b) Effect of glyphosate treatment on lignifications of fibres of some flax cultivars. Ann Appl Biol 108:114–115Google Scholar
  41. Sharma HSS, Lefevre J, Boucaud J (1992) Role of microbial enzymes during retting and their effect on fibre characteristics. In: Sharma HSS (ed) The biology and processing of flax. M publications, Belfast, pp 199–212Google Scholar
  42. Sharma HSS, Van Sumere CF (1992) In: Sharma HSS (ed) The biology and processing of flax, M publications, Belfast, 576 ppGoogle Scholar
  43. Song J, Weon HY, Yoon SH, Park DS, Go SJ, Suh JW (2001) Phylogenetic diversity of thermophilic actinomycetes and Thermoactinomyces spp. isolated from mushroom composts in Korea based on 16S rRNA gene sequence analysis. FEMS Microbiol Lett 202:97–102CrossRefPubMedGoogle Scholar
  44. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849CrossRefGoogle Scholar
  45. Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins MD, Doré J (1999) Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 65:4799–4807PubMedCentralPubMedGoogle Scholar
  46. Tahir P, Ahmed AB, SaifulAzry SOA, Ahmed Z (2011) Retting process of some bast plant fibres and its effect on fibre quality: a review. Bioresources 6:5260–5281Google Scholar
  47. Tamburini E, Gordillo Leon A, Perito B, Di Candilo M, Mastromei G (2004) Exploitation of bacterial pectinolytic strains for improvement of hemp water retting. Euphytica 140:47–54CrossRefGoogle Scholar
  48. Tian JH, Pourcher AM, Bouchez T, Gelhaye E, Peu P (2014) Occurrence of lignin degradation genotypes and phenotypes among prokaryotes. Appl Microbiol Biotechnol 98:9527–9544CrossRefPubMedGoogle Scholar
  49. Vignon MR, Garcia-Jaldon C (1996) Structural features of the pectic polysaccharides isolated from retted hemp bast fibres. Carbohydr Res 296:249–260CrossRefPubMedGoogle Scholar
  50. Visi DK, D’Souza N, Ayre BG, Webber Iii CL, Allen MS (2013) Investigation of the bacterial retting community of kenaf (Hibiscus cannabinus) under different conditions using next-generation semiconductor sequencing. J Ind Microbiol Biotechnol 40:465–475CrossRefPubMedGoogle Scholar
  51. Wright ES, Yilmaz LS, Noguera DR (2012) Decipher, a search-based approach to chimera identification for 16S rRNA sequences. Appl Environ Microbiol 78:717–725CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Alexandra Ribeiro
    • 1
    • 2
  • Philippe Pochart
    • 1
    • 3
  • Arnaud Day
    • 2
  • Sarah Mennuni
    • 2
  • Pierre Bono
    • 2
  • Jean-Luc Baret
    • 4
  • Jean-Louis Spadoni
    • 5
  • Irène Mangin
    • 1
    • 3
  1. 1.Microbial Ecology LaboratoryConservatoire National des Arts et MétiersParisFrance
  2. 2.Fibres Recherche Développement®Technopole de l’Aube en ChampagneBréviandesFrance
  3. 3.EA 4065 Cnam-Paris Descartes, Faculté des Sciences Pharmaceutiques et BiologiquesUniversité Paris DescartesParis Cedex 06France
  4. 4.Laboratoire Industries agroalimentairesConservatoire National des Arts et MétiersParisFrance
  5. 5.Laboratoire Génomique, Bioinformatique et ApplicationsConservatoire National des Arts et MétiersParisFrance

Personalised recommendations