3.1. Colour Change
The colour change of the stems and the fibres issued from different retting times for examined harvest periods (BF, EF and SM) was visually evaluated. As can be observed in the photograph (Figure 4
), the colour varies with plant growth and also during field retting. For BF and EF, the colour of the unretted samples was light green, contrarily to the stems and fibres harvested at SM that were transformed into yellow. This variation of the color from green to yellow after maturity is presumably due to the change in the light depth penetration in the photosynthetic tissues. Indeed, the chlorophyll that is responsible for the green colour was decomposed with the partial retention of carotenoids that emit a yellow colour [25
During the retting of samples harvested at BF and EF, the colour changed between light green for unretted samples (R0), yellow for low retted samples (R1 to R3), yellow with the existence of grey fibres for medium retted samples (R4 and R5) and dark grey for highly retted samples (R9). In contrast, for the samples of SM, the colour only changed from yellow (R0) to dark grey (R5). The fibres harvested at BF and EF required 9 weeks to be completely dark grey, while only 3 weeks were needed for the fibres harvested at SM. This indicates that the retting of the samples collected at SM was fast due to weather conditions (rainy retting period) and the aging of the stems at this stage. In contrast, as described in Section 2.1.2
, the retting of stems harvested at BF and EF was performed under dry weather conditions, allowing the observation of a slow and gradual colour transition during retting of these periods.
This colour transition is typical during field retting, even for other natural fibres such as flax fibres [11
]. The colour variation from yellow to grey for the fibres is related to the development of microbial communities (fungal and bacteria [29
] at stem surface. In addition, during field retting, black spots appeared and increased on the stems surface until the colour of the stems became black. The ESEM images (Figure 5
) of the stems harvested at SM (for example) reveal that the black spots are attributed to the development of microbial communities at the stem surface during field retting. The microbial communities would start to colonize the surface of stems harvested at SM after 1 week of retting (R1SM) and gradually cover the entire stems after 3 weeks (R3SM). However, for BF and EF periods, large coverage of the stem surface by microorganisms was observed after 9 weeks of retting [22
3.2. Biochemical Composition
The evolution of relative biochemical composition as a function of retting duration is presented in Figure 6
for fibres harvested in the EF and SM period. The evolution for fibres harvested at BF growth stage was detailed in a previous paper [22
]. The results show that the biochemical variation depends on both the harvest period and the retting duration.
For all the harvest periods, a gradual increase of cellulose content could be observed during field retting. The cellulose increased from 63% to 77% after 9 retting weeks for fibres harvested at the BF period [22
], and from 68% to 75% after 9 retting weeks for fibres harvested at EF period, whereas it increased from 70% to 74% after 5 retting weeks for fibres harvested at SM period. In addition, an increase of the cellulose content can be noted from 63% to 68% then to 70% over the growth period from BF to EF and then to SM, respectively. As the quantification of cellulose content is relative, thus, its variation is related to the change of content of other components. The pectins content decreased during retting for fibres harvested at BF (from 7.8% for R0BF to 4.1% for R9BF) [22
], for those harvested at EF (from 7.5% for R0EF to 4.4% for R9EF) and finally, those harvested during the SM period (6.2% for R0SM to 3.9% for R5SM). Moreover, a decrease of the pectins content can be noted from 7.8% to 7.5%, then to 6.2% during the growth period from BF to EF then to SM, respectively. A slight reduction in hemicelluloses content was given in evidence with increasing of retting duration (14.1% to 11.6% between R0BF and R9BF, 15.7% to 14.9% between R0EF and R9EF and 16.0% to 14.5% between R0SM and R5SM). Nykter et al. [32
] used the same extraction method and found an identical tendency of these components after enzymatic treatments. The gradual degradation of pectins is consistent with data reported by Meijer et al. [33
] and Musialak et al. [34
] during field retting. As regards Liu et al. [13
], these authors pointed out an increase of cellulose content at early stage of field retting with a decrease of non-cellulosic components (pectins and hemicelluloses).
shows also a decrease in minerals (ash) and lipophilic extractives (waxes, fats, resins) content during the retting of fibres harvested during the BF [22
], EF and SM periods. Ash contents decreased from 4.2% for R0BF to 1.5% for R9BF, from 3.2% for R0EF to 1.5% for R9EF and from 2.3% for R0SM to 1.6% for R5SM. Lipophilic extractives contents decreased from 14.9% for R0BF to 4.1% for R9BF, from 8.6% for R0EF to 2.7% for R9EF and from 4.0% for R0SM to 2.6% for R5SM. Fibres harvested at BF and EF period have the highest content of these components compared to that of fibres harvested during the SM period. This might justify the long retting period of fibres harvested at the BF and EF growth stages, since the high presence of lipophilic extractives (waxes) and green fresh odour could be an obstacle to the retting process by reducing its efficiency, and thereby, increases its duration. Foulk et al. [35
] reported that the presence of wax on the cuticle forms a protective barrier against entry of infection agents into the plant. The lignin content increased during field retting time for fibres harvested at BF [22
], EF and SM periods (from 3.1% for R0BF to 5.1% for R9BF, from 3.9% for R0EF to 9.3% for R9EF and from 5.0% for R0SM to 8.0% for R5SM). This result is in agreement with Liu et al. [13
] and Placet et al. [12
], who reported that during field retting, the lignin content increased due to a lower rate degradation of lignin compared to the other components (lipophilic extractives, ash and carbohydrates) that are removed at the same time. Placet et al. [12
] suggested another explanation. Indeed, other phenolic or protein components evolved during retting might be also measured through the used Klason method. On the other hand, this increase could be related to the fibres’ extraction method from the stems. Indeed, the different parts of the stems (e.g., epidermis, xylem) may become weaker with the increase of the field retting time. Therefore, when fibres are extracted from the retted stems, micro-residuals xylem (shives) could be still bounded to the fibres and overestimate the real variation of lignin during retting treatment. This variation in biochemical composition is due to both biofilm growth (ESEM investigations) and the metabolic activity of microorganisms during hemp fibres retting [29
3.3. Cellulose Crystallinity
In addition to the biochemical analysis, the influence of field retting on the cellulose organization of fibres harvested during the EF and SM periods was characterized using X-ray diffraction. X-ray diffractograms of the fibres gathered at different retting duration for EF and SM periods were obtained after an XRD analysis (Figure 7
). All the samples exhibit three defined peaks that are located at 2θ diffraction angles of 14.8°, 16.2° and 22.6°, and which correspond to crystallographic planes of cellulose type I: (101),
) and (002), respectively. The XRD patterns show that the intensity of the crystalline peak rose with the field retting process for both fibres harvested during the EF and SM periods, as previously observed for fibres harvested during the BF period. When the crystalline cellulose content is high, peaks at around 14° and around 16° are quite separated. But when the fibres contain a higher amount of amorphous phase (e.g., for unretted fibres), these two peaks merged and appeared as one broad peak [36
]. Furthermore, in order to quantify these differences, the crystalline order index (CI) was determined from X-ray diffractograms using a deconvolution method described in the Materials and Methods Section. Table 2
presents CI values for hemp fibres harvested during the EF and SM periods showing a gradual increase with the field retting duration from 58% to 69% and from 64% to 73% for fibres harvested during the EF and SM periods, respectively. This was also noticed during field retting of hemp fibres harvested during the BF period with an increase from 53% to 73% [22
]. The high value of CI of unretted fibres harvested at SM (R0SM) compared that of unretted fibres (R0EF) is not surprising, since according to the results of the biochemical analysis, the cellulose content of R0SM is higher than that of R0EF. The increase in CI was also noticed by Li et al. [37
], who compared green hemp fibres, 1-week retted fibres and 2-week retted fibres using bag retting treatment. They found that the CI evolved from 66% for hemp green fibres to 85% for retted hemp fibres, respectively. Zafeiropoulos et al. [38
] reported an increase of CI from 65% to 72% after a field retting of flax fibres. This increase of CI could be explained by the degradation of non-cellulosic compounds during retting enabling packing of cellulose chains. Indeed, a high amount of amorphous constituents presented between the cellulose micro-fibrils causes disoriented areas, which could undesirably influence the crystallinity of the cellulose micro-fibrils. The change in cellulose fraction, and in cellulose structure, as well as its degree of crystallization, may have a direct impact on the mechanical properties [6
3.4. Thermal Stability
A thermogravimetric analysis (TGA) was carried out in order to assess the influence of field retting duration on the thermal performance of the fibres harvested at different hemp plant growth stages. Figure 8
shows the curves of TGA and DTGA obtained from different retting durations of fibres harvested during the EF and SM periods. Results for fibres harvested at BF growth stage were detailed in a previous paper [22
]. In general, there are three stages of decomposition in TGA curves (Figure 8
A,C). The initial weight loss at about 30–100 °C is due to the evaporation of the absorbed moisture. The second at about 230–260 °C is related to the decomposition of non-cellulosic components (pectins and hemicelluloses) and the third stage at 335 °C is attributed to the decomposition of major component of fibres (cellulose). This process of decomposition of the fibres is consistent with data reported in literature [12
In TGA curves, peaks are superimposed on a temperature scale, thus, by calculating the derivative of the weight loss as a function of temperature (Figure 8
B,D). The decomposition process of the fibres can be clearly observed in this case. From the peak with the highest intensity, it can be seen that the increase in the retting duration led to an increase of the decomposition temperature of the fibres, from 337 °C to 359 °C for R0BF and R9BF, from 323 °C to 357 °C for R0EF and R9EF and from 350 °C to 367 °C for R0SM and R5SM. This emphasized a higher thermal stability for high retted fibres, as confirmed by the literature [12
]. As the field retting of the fibres harvested at EF was long, the increase of the decomposition temperature of the fibres was gradual and slow. In contrast, for the fibres harvested at SM, the increase in the decomposition temperature of the fibres was rapid and high. In addition to the retting treatment, it can also be observed that the R0SM fibres displayed a higher decomposition temperature (350 °C) when compared to R0EF fibres (323 °C). The peak intensity (shoulder peak) of the pectins and hemicelluloses decreased as a function of the retting duration. This is related to their partial degradation during field retting. However, this peak is not visible when the hemp fibres are highly retted, indicating that non-cellulosic components were quasi-totally removed. The previously described increase in cellulose temperature decomposition can also be related to the removal of non-cellulosic components (amorphous materials). This phenomenon brings a higher structural order of cellulose with strong intramolecular and molecular hydrogen bonds that need a higher degradation temperature to be broken down [43
The evolution in plant morphology of hemp during three growth stages (BF, EF and SM) was investigated. Hemp stems contained different layers, as already described by number of authors [13
]. They are organized from the stem pith toward the surface by central woody core (xylem designed as X), cambium, cortex (including both primary -and secondary fibres noted PFB and SFB respectively) and epidermis (called E). During growth from the beginning of flowering to the maturity of the plant, a variation in the morphological characteristics of the plant occurs, but the same organization of layers is always observed. The diameters of the unretted stems harvested during the BF, EF and SM periods, and before retting were 5.7 ± 0.7 mm, 6.1 ± 0.5 mm and 6.5 ± 0.1 mm, respectively. This increase is associated to the variation of the layers thickness of different parts of the stem. This study shows that the morphological features of hemp stem depend on the harvest period (Figure 9
). During plant growth from BF to SM, the thickness of bast fibres and of xylem layers increased. This change is particularly related to the increase in secondary fibres layer (SFL) thickness from 20 µm for BF to 40 µm for SM, while no significant variation of the thickness of primary fibres layer (PFL) and epidermis layer (EL) were observed with the growth period. This result was already reported by Liu et al. [13
] and Mediavilla el al. [9
]. Tanja Schäfer. [46
] pointed out that dry weather conditions result in a higher presence of secondary fibres in the hemp stem.
The impact of field retting duration on the morphology of the hemp fibres was then qualitatively analyzed. Figure 10
shows the optical microscope micrographs of cross-sections of unretted (R0EF) and retted hemp stems (R3EF, R5EF and R9EF) collected at the end of flowering. When the hemp stem is unretted (R0EF), the structure of different layers of the stem is intact and well organized. The fibres are gathered in the form of a bundle and the lumen of the elementary fibres cannot be distinctly observed. Since that field retting period of stems harvested at EF was long and slow, no high difference could be observed after 3 weeks (R3EF) of retting at the level of bast fibre. In contrast, after 5 weeks of retting (R5EF), the structure of stem changed. The primary and secondary fibre bundles were separated into smaller fibres bundles, resulting in more open spaces between the fibre bundles. When the stems were highly retted (R9EF), the structure of the stem was affected by field retting. The epidermis layer was deformed and removed. The bundles of fibres were completely separated into elementary fibres (EF) and their lumen (L) is clearly visible. A similar evolution of the morphology of the hemp fibres during the field retting process was observed in our recent work [22
] for stems harvested at BF. Overall, whatever the growth period, the same evolution of the morphology of the hemp fibres during field retting process is highlighted. The bast fibre bundles are separated (i) from the central woody core and epidermis (ii) and into smaller bundles or individual fibres.
This change in morphology during retting is due to the microorganisms’ activities that would allow the removal of intercellular cementing components (pectins and lipids extractives) in agreement with the biochemical analyses. This separation of the fibres into smaller bundles or elementary fibres during field retting would have a positive effect on the mechanical properties of hemp fibres as reinforcements in composites [11
3.6. Tensile Properties
In order to evaluate the influence of field retting on the hemp fibres harvested at different growth stages (BF, EF and SM), the mechanical properties of the fibre bundles were characterized by micro-tensile tests. As shown in Figure 11
A, the fibre bundle diameters selected for each batch were approximately between 100 and 240 µm with a median value at about 160 µm so that it was possible to compare relatively the results of micro-tensile tests. Figure 11
A–D presents the tensile properties (tensile strength, Young’s modulus and strain at failure) of the fibre bundles extracted at the different growth stages and retted at different times.
As concerns the influence of the growth stage, an increase of all the mechanical characteristics (considering median values) with plant growth can be observed. Indeed the tensile strength increased from 174 MPa for the R0BF to 331 MPa for R0EF and then reached up to 352 MPa for R0SM. Fibres harvested at BF (R0BF) have a lower tensile strength than that of the fibres harvested during the EF and SM periods, while no significant difference (p
> 0.05) is observed between R0EF and R0SM. Young’s modulus increased significantly from 8 GPa for R0BF to 13 GPa for R0EF and R0SM. A similar trend is observed for strain at break. It increased significantly from 2.4% to 3.2% and 3.4% for fibres harvested during the BF, EF and SM periods, respectively. According to these results, it can be concluded that the main improvement of tensile properties occurred between the fibres extracted at the end of flowering (R0EF) and the beginning of flowering (R0BF), as just a slight increase was observed for fibres collected at the seed maturity period (R0SM). This change in tensile properties during plant growth might be due to the variation in the biochemical composition, fibres morphology and fibres extraction. Indeed, the increase of cellulose fraction with growth stage could play a key role in increasing mechanical performance [6
]. Moreover, Goudenhooft et al. [10
] highlighted that the morphology of the fibres could also impact the mechanical performance. They reported an increase of the mechanical properties of flax fibres during plant development. Another explanation could be attributed to the fibres’ damage by the manual decortication of the fibres from the stems. It was visually noticed that the hand extraction of the fibres after removing the epidermis was easier for fibres collected during the EF and SM periods compared to BF. This means that there was less generation of micro-defects during extraction of the fibres harvested at EF and SM. Keller et al. [9
] reported that harvest at seed maturity led to easier decortication and a high tensile strength of hemp fibres.
In addition to this comparison of tensile properties of the initial state of the fibres after the BF, EF and SM harvest periods, a variation in the mechanical performance during retting treatment of hemp fibres was also observed for these selected growth stage. The tensile strength of the hemp fibres of BF increased from 174 MPa for unretted fibres (R0BF) to 342 MPa after 5 weeks of retting (R5BF), and then a slight decrease to 324 MPa was observed after 9 weeks of field retting (R9BF) (extended field retting). Young’s modulus of increased significantly (p < 0.05) from 8 GPa for unretted hemp fibres (R0BF) to around 12 GPa for retted hemp fibres (R5BF and R9BF). Likewise, the strain at failure increased significantly (p < 0.01) from 2.4% for unretted fibres (R0BF) to 2.8%, 3.4%, and 3.3% for retted hemp fibres R3BF, R5BF and R9BF, respectively.
The trend evolution of the tensile properties of the fibres harvested at EF is identical to those of BF, although some interesting differences are noted. In particular, the fibres harvested at EF exhibited higher mechanical properties during retting. The tensile strength, Young’s modulus and strain at failure of fibres harvested at EF increased, respectively, from 331 MPa, 13 GPa and 3.2% for R0EF to 487 MPa, 17 GPa and 3.4% for R5EF and then decreased significantly to 323 MPa, 13 GPa and 2.8%, respectively, for R9EF. Concerning the results of the fibres harvested at SM, no significant difference can be noticed for the tensile strength, even though it tended to decrease from 352 MPa for R0SM to 282 MPa for R5SM. No apparent change could be observed for Young’s modulus, contrarily to the strain of failure that decreased gradually and significantly from 3.5% for R0SM to 2.4% for R5SM. Whatever the plant growth stage, a maximum of tensile properties is reached after five weeks and then reduced with an extended of field retting.
During field retting of all the examined harvest periods, the cellulose fraction and the cellulose chain packing order were improved due to the removal of non-cellulosic materials, which could bring better tensile performances [39
]. However, since hemp fibres can be seen as a natural composite of cellulose microfibrils and a matrix of non-cellulosic components, the mechanical properties are not only governed by cellulose and crystallinity index, but also by the coherence between the cellulose and non-cellulosic components [48
]. Therefore, a high degradation of non-cellulosic components with extended retting duration (over-field retting), might override the influence of the increased cellulose and crystallinity and thereby, result in lower tensile properties. In addition, generally, with increasing retting duration, the cementing compounds that bind different parts of the stem are gradually removed by microorganisms. This allows an easier hand separation of the bast fibres from the ligneous shives and limits the engendering of the micro-defects in the fibres.
These results clearly indicate that the mechanical properties of the hemp fibres depend on the plant growth period and the retting duration which is governed by the weather conditions. The retting periods of fibres harvested at BF and EF were long because they were carried out under dry weather conditions, contrarily to the retting period of fibres harvested at SM that performed under rainy weather conditions. Therefore, a long period (5 weeks) is required to obtain the highest mechanical properties of fibres harvested at BF and EF. However, the retting of fibres harvested at SM has to be done in a short period (around 1 week) in order to avoid over-retting treatment. In a recent work, Placet et al. [49
], compared three times (10, 39 and 75 days) of field retting of hemp fibres and found that the mechanical properties of single hemp fibres increased at the early stage and then decreased with prolonged field retting. Liu et al. [13
] also showed that a negative effect of field retting occurred with extended retting duration due to the high rate degradation of cellulose by microorganisms. To this end, according to the results obtained in this study, in order to avoid an under- and over-retting treatment, it would be judicious to choose the end of flowering period for two reasons: (i) the tensile properties of the initial state of the fibres after harvesting were high (ii) the retting period coincides with two seasons (summer and autumn) which allows the mastering of retting mechanisms.