The existence of spun and colored flax fibers dating from the Upper Paleolithic suggests that man has long exploited the biotechnological process known as retting to extract these fibers from the flax plant for textile production [1, 2]. In the case of dew-retting, the complex microbiological process is achieved directly on the soil surface of the field [3, 4]. Despite many studies of this process to evaluate the degree of retting [5], relatively little is known about (i) the composition and the dynamics of the microflora population during retting, (ii) the spatial-temporal effect on the microbiome and (iii) the composition and the evolution of Carbohydrate Active enZymes degrading plant cell walls [6]. To improve our understanding of dew-retting, we used a high throughput sequencing (HTS) metabarcoding approach (16S rRNA and ITS regions, Illumina Miseq system) to analyze the composition and dynamics of the rhizosphere (soil) and caulosphere (stem) microbial populations during this process. This study was conducted within the framework of the collaborative French ‘Future project’ ‘StructuratIoN de la filière Fibres techniques d’OrigiNe végétale pour usages matérIaux’ (SINFONI). Our results allowed us to (i) report the first exhaustive HTS microbial inventory focusing on both bacteria (16S rRNA) and fungi (Internal Transcript Spacer 2), (ii) evaluate the impact of a number of different parameters likely to affect microbiome structural variability and retting efficiency and (iii) to ascertain the bacterial and fungal groups responsible for the observed community differentiation in the caulosphere. In addition, we also used PICRUSt to predict potential bacterial enzymatic functions related to carbohydrate degradation and FUNGuild to determine fungal trophic modes [7, 8].
[1] Gübitz, G.M. & Cavaco-Paulo (2001) Journal of biotechnology, 89(2–3), pp.89–90.
[2] Kvavadze, E. et al. (2009) Science, 325(5946), pp.1359–1359
[3] Md. Tahir, P. et al. (2011) BioResources, 6(4), pp.5260–5281
[4] Akin, D.E., (2013) ISRN Biotechnology, 2013, p.23
[5] Martin, N. et al. (2013) Industrial Crops and Products, 49, pp.755–767
[6] Lombard, V. et al. (2014) Nucleic acids research, 42(Database issue), pp.D490-5 [7] Langille, M.G. et al. (2013) Nat Biotechnol, 31(9), pp.814–821
[8] Nguyen, N.H. et al. (2016) Fungal Ecology, 20, pp.241–248.

The existence of spun and colored flax fibers dating from the Upper Paleolithic suggests that man has long exploited the biotechnological natural process known as dew-retting to extract these fibers from the flax plant for textile production [1,2]. This process is achieved directly on the soil surface of the field [3,4]. Despite many studies of this process to evaluate the degree of retting [5], relatively little is known about (i) the composition and the evolution of the microflora population during retting, (ii) the kinetics of the microbial communities colonizing the plant material and (iii) the composition and the evolution of Carbohydrate Active enZymes degrading plant cell walls [6]. To improve our understanding of dew-retting, we first used a metabarcoding approach to identify the membership and structure of the microbial communities (focusing on bacteria and fungi). This approach also allowed us to identify (i) some potential bacterial major enzymatic functions related to carbohydrate degradation based on functional prediction using PICRUSt (http://picrust.github.io/picrust/) and (ii) a strong pattern of fungal trophic modes [7,8]. In a second step we developed a metatranscriptomic approach to access of the evolution of the exogenous (soil) and endogenous (plant) enzymatic arsenal potentially across the Tree of Life, involved in the degradation of carbohydrate and aromatic substances in decomposing plant matter. The methodology used to explore microbial and enzymatic diversity using High Throughput Sequencing (Illumina system) will be described. We will then correlate colonization complexity dynamics to the progress in plant cell wall degradation. Finally, the microbial ecology of the retting process will be compared to other natural plant material (e.g. forest litter) degradation processes.
Acknowledgements
This work is funded within the framework of the collaborative French ‘Future project’ SINFONI.
References
[1] Roland, J.-C., Mosiniak, M. & Roland, D., 1995. Dynamique du positionnement de la cellulose dans les parois des fibres textiles du lin ( Linum usitatissimum ). Acta Botanica Gallica, 142(5), pp.463–484.
[2] Kvavadze, E. et al., 2009. 30,000-Year-Old Wild Flax Fibers. Science, 325(5946), pp.1359–1359.
[3] Md. Tahir, P. et al., 2011. Retting process of some bast plant fibres and its effect on fibre quality: A review. BioResources, 6(4), pp.5260–5281.
[4] Akin, D.E., 2013. Linen most useful: Perspectives on structure, chemistry, and enzymes for retting flax. ISRN Biotechnology, 2013, p.23.
[5] Martin, N. et al., 2013. Influence of the degree of retting of flax fibers on the tensile properties of single fibers and short fiber/polypropylene composites. Industrial Crops and Products, 49, pp.755–767.
[6] Lombard, V. et al., 2014. The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic acids research, 42(Database issue), pp.D490-5.
[7] Langille, M.G. et al., 2013. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol, 31(9), pp.814–821.
[8] Nguyen, N.H. et al., 2016. FUNGuild: An open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecology, 20, pp.241–248.

The first step in the industrial transformation of flax stems into fibers is achieved by dew-retting on the soil surface (Tahir et al., 2011 and Akin et al., 2013). Despite many studies of this process relatively little is known about i) the composition and the evolution of the microflora population, ii) the kinetics of microbial colonization of plant material and iii) the composition and the evolution of the plant cell wall-degrading enzymes targeted to the different CAZy families (Lombard et al., 2014).
In this work, we report the first exhaustive microbial inventory of dew retting. We conducted a targeted-metagenomics approach to identify for the first time the composition of the microbial communities (focusing on bacteria and fungi) involved in flax dew-retting. This approach has also allowed us to identify some potential major enzymatic functions related to cell wall degradation based on functional prediction using the bioinformatic software PICRUSt (Langille et al., 2013). In a second step we developed a metatranscriptomic approach to access the exogenous (soil) and endogenous (plant) enzymatic arsenal potentially involved in degrading cell wall polysaccharides during dew-retting in flax.
The methodology used to explore microbial and enzymatic diversity using the Next Generation of Sequencing (Illumina system) will be described. We will then correlate colonization dynamics to plant cell wall degradation. Finally, the microbial ecology of the retting process will be compared to the degradation process of other plant-based material (e.g. forest litter).
This work is funded within the framework of the collaborative French ‘Future project’ SINFONI.

The first step in the industrial transformation of flax stems into industrial fibers used in textiles or composite materials is achieved by dew-retting on the soil surface. At harvest, the flax stems are ‘pulled’ (up-rooted) and laid down directly on the soil in swaths (strips). Subsequent exposure to alternating periods of rain and heat combined with the development and the action of soil microflora favor the separation of cellulosic bast fibers from the stems via the hydrolysis of cell wall polymers (hemicelluloses, pectins). Despite many studies of this process (Akin, 2013; Henriksson, & Akin, 1997; Martin et al., 2013) relatively little is known about i) the composition of the microflora population, ii) the kinetics of microbial colonization of plant material and iii) the evolution of the microbial population in the soil. As an example of biological processing involving microbial activities, dew retting represents a convenient model to study the microbial dynamics of plant colonization and degradation of cell wall components.
In order to study this biological process, we undertook a targeted-metagenomics approach. A metagenomic pipeline including sample preparation, marker amplification, raw sequence and data analyses was built. We evaluated and compared different protocols and tools such as mothur (Schloss et al., 2009), qiime (Caporaso et al., 2010), PIPITS (Gweon et al., 2015) and clustering tools such as swarm (Mahé et al., 2014). The resulting pipeline analyses and homemade tools used to redesign primers so as to specifically amplify bacterial rRNA genes from complex samples including plants are detailed. Analyses are completed by functional predictions of enzymatic activity using an adaptation of PiCRUSt (Langille et al., 2013) software for the CAZy database (Lombard et al., 2014).
As a conclusion and proof of pipeline efficiency, we report the first exhaustive microbial inventory of dew-retting and its evolution during the process obtained by using a targeted-metagenomics approach. Potential enzymatic functions related to cell wall degradation based on functional prediction using the bioinformatic software PICRUSt are listed and used to establish a potential chronology of cell wall polymer degradation.
This work is funded within the framework of the collaborative French ‘Future project’ SINFONI. Christophe Djemiel thanks the ‘Haut de France region’, and OSEO for their financial support. The authors thank the Genomic platform of Genopole Toulouse Midi Pyrénées where sequencing was performed.
References :
Akin, D. E. (2013). Linen most useful: Perspectives on structure, chemistry, and enzymes for retting flax. ISRN Biotechnology, 2013, 23. http://doi.org/http://dx.doi.org/10.5402/2013/186534
Henriksson, G., & Akin, D. (1997). Identification and retting efficiencies of fungi isolated from dew-retted flax in the United States and europe. Applied and Environmental Microbiology. Retrieved from http://aem.asm.org/content/63/10/3950.short
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. Industrial Crops and Products, 49, 755–767. http://doi.org/10.1016/j.indcrop.2013.06.012
Schloss, P. D., Westcott, S. L., Ryabin, T., Hall, J. R., Hartmann, M., Hollister, E. B., … Weber, C. F. (2009). Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Applied and Environmental Microbiology, 75(23), 7537–41. http://doi.org/10.1128/AEM.01541-09
Caporaso, J. G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F. D., Costello, E. K., … Knight, R. (2010). QIIME allows analysis of high-throughput community sequencing data. Nature Publishing Group, 7(5), 335–336. http://doi.org/10.1038/nmeth0510-335
Gweon, H. S., Oliver, A., Taylor, J., Booth, T., Gibbs, M., Read, D. S., … Schonrogge, K. (2015). PIPITS: An automated pipeline for analyses of fungal internal transcribed spacer sequences from the Illumina sequencing platform. Methods in Ecology and Evolution, 6(8), 973–980. http://doi.org/10.1111/2041-210X.12399
Mahé, F., Rognes, T., Quince, C., de Vargas, C., & Dunthorn, M. (2014). Swarm: robust and fast clustering method for amplicon-based studies. PeerJ, 2, e593. http://doi.org/10.7717/peerj.593
Langille, M. G., Zaneveld, J., Caporaso, J. G., McDonald, D., Knights, D., Reyes, J. A., … Huttenhower, C. (2013). Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nat Biotechnol, 31(9), 814–821. http://doi.org/10.1038/nbt.2676
Lombard, V., Golaconda Ramulu, H., Drula, E., Coutinho, P. M., & Henrissat, B. (2014). The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Research, 42(Database issue), D490–5. http://doi.org/10.1093/nar/gkt1178