A Comparative Study of the Effect of Field Retting Time on the Properties

Article A Comparative Study of the Eﬀect of Field Retting Time on the Properties of Hemp Fibres Harvested at Diﬀerent Growth Stages

Brahim Mazian 1,2,*, Anne Bergeret 1,*, Jean-Charles Benezet 1 and Luc Malhautier 2

1 Centre des Matériaux des Mines d’Alès, IMT Mines Alès, Université de Montpellier, 6 avenue de Clavières, 30319 Alès Cedex, France; [email protected] 2 Laboratoire du Génie de l’Environnement Industriel, IMT Mines Alès, Université de Montpellier, 6 avenue de Clavières, 30319 Alès Cedex, France; [email protected] * Corresponding: [email protected] (B.M.); [email protected] (A.B.)

Received: 25 October 2019; Accepted: 5 December 2019; Published: 7 December 2019

Abstract: In this study, the comparison of field retting of hemp fibres harvested at different growth stages (beginning and end of flowering, seed maturity) was studied. Regardless of the harvest period, identical evolution of the fibres’ properties was observed during retting. The main difference is the kinetics of this transformation, which depend on weather conditions and the initial state of the fibres after harvesting. Retting leads to a change in colour of the stems and fibres, an increase of the cellulose fraction and a gradual improvement of the fibres’ thermal stability, in relation with a decrease in the non-cellulosic materials. This process induces fibre bundle separation into elementary fibres. A long period (5 weeks) is required for getting the highest mechanical properties of fibres harvested at the beginning and the end of flowering. However, the retting of fibres harvested at seed maturity has to be performed in a short period (1 week) in order to avoid over-retting treatment. If the fibres are over-retted, their quality decreases in terms of structure and mechanical properties.

Keywords: hemp fibre; plant growth; retting temporal dynamics; mechanical properties; thermal stability

1. Introduction Based on environmental concern, the interest in using the cellulosic fibres has increased in high grade composites in recent years. Plant fibres issued from hemp (Cannabis sativa L.) are part of these cellulosic fibres which could be used as an alternative of conventional fibres (e.g., glass and synthetic fibres) for manufacturing industry of low cost, low weight composite materials. This attraction of hemp fibres as reinforcement agents in composite is related to their high specific mechanical properties [1,2], their biodegradability [3,4], as well as their low-density [5]. However, there are a number of problems associated with the variation of the quality of these fibres in term of the morphology, chemical composition and physical properties, which results in a large variation in the mechanical properties of the fibres that can impact the quality of final composite material [6]. Many parameters are involved, such as the variety [7], the growth conditions (ground, weather) [8], the plant growth stage [9,10], and the traditional extraction and separation processes, e.g., retting [11–13]. Hemp fibre can be considered as a complex natural composite of cellulose microfibrils and matrix of amorphous polysaccharide, mainly composed of pectins and hemicelluloses. Indeed, the microfibrils of cellulose are embedded in a matrix of pectins and hemicelluloses, forming the different cell wall layers of an elementary fibre [2,14]. The elementary fibres are assembled together through their pectin middle lamella to form bundles of fibres [15]. For using these fibres in material composite, a separation of bundle of fibres to individual fibres or smaller fibres bundles is necessary for improving

Fibers 2019, 7, 108; doi:10.3390/fib7120108 www.mdpi.com/journal/fibers Fibers 2019, 7, 108 2 of 18 interfacial bonding between fibres and matrix, as well as for enhancing the mechanical properties of final composite [11,16,17]. For that purpose, a traditional treatment called retting process is usually used to ease the separation of the fibres. In Europe, the most widely used treatment is field retting (also known as dew-retting) because it is inexpensive and easy to be applied [18,19]. This treatment consists of cutting and leaving the hemp plants on the field so the microorganisms (fungi and bacteria) attack and colonize the stems, producing a wide range of enzymes, such as pectinolytic that lead to the removal of the components in the middle lamella (pectic substance) and allows the cortex fibres to be progressively separated [20–22]. Therefore, the field retting is a crucial step for the production of hemp fibres, but it is empirically realized in the field due to its dependence on the weather conditions (temperature, humidity) which could cause problems of the inconsistency of the hemp fibres quality. However, if this process is well controlled, much higher quality with more uniform fibres could be produced. Hemp seed exploitation pushes the farmers to harvest the hemp plants during the seed maturity period. For this, the field retting is commonly performed at this step of plant development, which coincides with the autumn period. During this period (mainly with heavy rainfall), the control of the fibres’ quality is sometimes not easy and over retting can be rapidly reached. Placet et al. [12] reported that when the weather conditions did not allow for collecting the stems in time during retting, the stems undergo an unwanted and uncontrolled retting. In addition, they found after the comparison between unretted and 5 weeks-retted fibres that the prolongation of field retting under a very rainy retting period caused over-retted fibres with lower quality in terms of mechanical properties. In contrast, when the stems are retted in summer weather conditions, a long period of retting is required [13,22]. Thus, understanding the impact of field retting treatment on the fibres harvested at different initial states of hemp plant development is needed for getting composite materials with high performance. Therefore, the purpose of this study was to compare the effect of the field retting on the properties of the fibres harvested at different growth stages under different conditions. Both qualitative and quantitative experimental techniques were used to survey the temporal dynamic of hemp fibres’ composition, microstructure, thermal and mechanical properties during retting of fibres.

2. Materials and Methods

2.1. Raw Material

2.1.1. Cultivation and Sampling Hemp (Cannabis sativa L., Cultivar ‘Santhica 27’) was sown at rate of 35 kg/ha on 6 May 2016 in the south of France (N 44.130673◦, E 4.315895◦) by CIVAM Chanvre Gardois [22]. The hemp plants were harvested manually using a shear at three growth stages (beginning of flowering (BF), end of flowering (EF), seed maturity (SM)) (Figure1). For each harvest period, at least 700 plants were harvested. The stems’ length during BF, EF and SM were 1.71 0.09 m, 1.83 0.10 m and ± ± 1.84 0.09 m, respectively. After each harvest period, the plants were spread out in the field for retting, ± and then a weekly collection of retted stems was performed. Table1 presents the retting duration and corresponding samples for each selected growth stage. Since morphological feature, chemical composition and mechanical properties depend on hemp stem sections [1,23,24], only the middle part of the stem was investigated [22] in order to limit the dispersion of the results. Fibers 2019, 7, 108 3 of 18 Fibers 2018, 6, x FOR PEER REVIEW 3 of 19

200

180

160

140

120

100 beginning of end of 80 flowering flowering seed maturity

Stem length (cm) length Stem (EF) 60 (BF) (SM)

40 25th July 16th August 23th September

Date (day)

Figure 1. Hemp plant growth from 6 May 2016 to 23 September 2016. Figure 1. Hemp plant growth from 6 May 2016 to 23 September 2016. Table 1. Sample names of different retting duration for different harvest periods (BF, EF and SM). Table 1. Sample names of different retting duration for different harvest periods (BF, EF and SM). Retting Time 0 1 2 3 4 5 6 7 9 Retting(Week) Time 0 1 2 3 4 5 6 7 9 (Week) R0BF R1BF R2BF R3BF R4BF R5BF - - R9BF Samples R0EFR0BF R1EF R1BF R2EFR2BF R3EFR3BF R4BF - R5EFR5BF - R7EF- R9BF R9EF name Samples nameR0SM R0EF R1SM R1EF R2SMR2EF R3SMR3EF R4SM- R5SMR5EF R6SM- R7EF - R9EF - R0SM R1SM R2SM R3SM R4SM R5SM R6SM - - 2.1.2. Weather Conditions 2.1.2. Weather Conditions A monitoring of temperature and relative humidity during retting of the stems harvested at diffAerent monitoring growth stagesof temperature was carried and out relative in the humidity field using during three hygro-boutonretting of the stems sensors harvested (Progesplus, at differentCarquefou, growth France) stages that was were carried directly out put in the in contact field using with three the soil. hygro In addition,-bouton sensors daily average (Progesplus, rainfall Carquefou,data were France) acquired that from were M édirectlytéo France put (weatherin contact station with the located soil. In at addition, Méjannes-le-Clap, daily average France rainfall (GPS: dataN44.221944 were acquired◦, E4.344722 from◦ )).Météo As can France be seen (weather in Figure station2, the located weather at conditions Méjannes were-le-Clap, not similarFrance during(GPS: N44.221944°,retting for each E4.344722°)). harvest period. As can The be retting seen ofin the Figure stems 2, harvested the weather at BF conditions was mainly were carried not out similar during duringsummer retting (25 Julyfor each to 26 har Septembervest period. 2016), The whereas retting theof the stems stems harvested harvested at EFat BF were was retted mainly during carried two outseasons during (summer summer and (25 autumnJuly to 26 (16 September August to 2016), 17 October whereas 2016)). the stems The rettingharvested of the at stemsEF were harvested retted duringat SM two was seasons performed (summer during and autumn autumn (23 (16 September August to to 17 7 October November 2016)). 2016). The Theretting weather of the was stems hot harvestedduring retting at SM for w theas performed stems harvested during at autumn BF. Indeed, (23 theSeptember average maximumto 7 November and minimum 2016). The temperature weather wasbetween hot during the 1st retting and the for 6ththe weekstems were harvested 47 ◦C at and BF. 17 Indeed,◦C, respectively, the average while maximum during and two minimum last weeks temperatureof this period, between they decreasedthe 1st and to the 26 6th◦C andweek 14 were◦C,respectively. 47 °C and 17Between °C, respectively, the 1st and while the during 4th week two of lastretting weeks of of the this stems period, harvested they decreased at EF, the to weather 26 °C and was 14 also °C, hot, respectively. with an average Between maximum the 1st and temperature the 4th weekof 49 of◦C retting and an of average the stems minimum harvested temperature at EF, the ofweather 16 ◦C. Fromwas also the hot 4th, weekwith toan theaverage end of maximum the retting temperatureperiod of EF, of the 49 temperature°C and an average decreased minimum with an temperature average maximum of 16 °C. and From minimum the 4th of week 23 ◦ Cto and the 13end◦C, ofrespectively. the retting period Regarding of EF, the the stems temperature harvested decreased at SM, the with retting an average of this periodmaximum was and performed minimum in coolof 23weather, °C and with13 °C, an respectively. average maximum Regarding temperature the stems of harvested 17 ◦C and at an SM, average the retting minimum of this temperature period was of performed10 ◦C. The in distribution cool weather, of rainfall with and an humidity average wasmaximum not homogeneous temperature during of 17 the °C retting and an period average of the minimumstems harvested temperature at BF of and 10 EF,°C. contrarily The distribution to the retting of rainfall period and of humidity stems of SM.wasThe not averagehomogeneous relative duringhumidity the duringretting rettingperiod ofof the the stems stems harvested harvested at at BF, BF EF and and EF, SM co wasntrar 62%,ily 64%to the and retting 75%, respectively.period of stemsDuring of SM. retting The of average the stems relative harvested humidity at BF during and EF, retting there of was the little stems rainfall harvested during at 9BF, weeks. EF and Indeed, SM wasbetween 62%, 64% the 1stand and 75%, 6th respectively. week of retting, During the retting sum ofof rainfallthe stems was harvested 32 mm forat BF BF and and EF, 95 there mm forwas EF, littlewhile, rainfall after during the 7th 9 week weeks. until Indeed, last sample between collection, the 1st and it was 6th higher,week of 82 retting, mm for the BF sum and of 162 rainfall mm forwas EF. 32Concerning, mm for BF and the retting95 mm periodfor EF, ofwhile, the stemsafter the harvested 7th week at until SM, thelast rainfall sample wascollection, more homogeneously it was higher, 82distributed. mm for BF Thereand 162 was mm rainfall for EF. almost Concerning, each week the throughoutretting period the of retting the stems period. harvested The sum at ofSM, rainfall the rainfallduring was the more 6 weeks homogeneously of retting period distributed. of the stem There of SM was is 215rainfall mm. almost each week throughout the retting period. The sum of rainfall during the 6 weeks of retting period of the stem of SM is 215 mm. Fibers 2019, 7, 108 4 of 18 Fibers 2018, 6, x FOR PEER REVIEW 4 of 19

60 Max temperature 50

Min temperature C) 40 30 20 10

Temperature( 0 -10 80 100 70 90 80 60 70 (%) 50 60 40 50

40 humidity 30 Rainfall

Rainfall(mm) 30 20 Relative humidity 20 10 10 Relative 0 0

R0 BF R9

R0 EF R9

R0 SM R6

Figure 2.2. DailyDaily minimumminimum andand maximummaximum temperature, temperature, relative relative humidity humidity and and rainfall rainfall during during retting retting of theof the stems stems harvested harvested at diatff differenterent growth growth stages stages (BF, (BF, EF andEF and SM). SM). 2.2. Experimental Methods 2.2. Experimental Methods 2.2.1. Visual Aspect 2.2.1. Visual Aspect The colour change of the stems and fibres issued from different retting durations for each harvest periodThe (BF, colo EFur and change SM) wasof the visually stems evaluated. and fibres issued The extraction from different of the fibresretting from durations the stems for each was manuallyharvest period performed (BF, EF using and aSM) razor was blade visually and flatevaluated. files. The extraction of the fibres from the stems was manually performed using a razor blade and flat files. 2.2.2. Microscopic Observations 2.2.2. Microscopic Observations The surface change of stems during retting was observed through scanning electron microscopy (ESEM)The using surface an environmental change of stems scanning during electron retting microscope was Quantaobserved FEG through 200 (FEI, scanning Merignac, electron France) withmicroscopy a magnification (ESEM) ofusing x500 an and environmental x1000. scanning electron microscope Quanta FEG 200 (FEI, Merignac,The eff France)ect of the with plant a magnification development stageof x500 (BF, and EF x and1000. SM) and retting duration on the morphology of theThe stem effect were of examined the plant using development the optical stage microscopy (BF, EF (Leica and Laborlux SM) and 11 retting POL S) durat equippedion on with the amorphology 1600 1200 of pixels the stem mono-CCD were examined Sony digital using camera. the optical The microscopy cross sections (Leica of Laborlux the stems 11 were POL cut S) × manually,equipped with then coateda 1600 × in 1200 an epoxy pixels resin mono (Geofix-CCD ®Sonyresin digital (ESCIL, camera. Chassieu, The France))cross sections and subsequently of the stems polishedwere cut using manually, different then fine coated polishing in an papers epoxy (600, resin 2400 (Geofix and® 4000resin grade) (ESCIL, to obtain Chassieu, smooth France)) samples. and Thesubsequently Archimed polished® software using was different used in order fine polishingto record digital papers images (600, 2400 (Figure and3 ) 4000 to realize grade) qualitative to obtain andsmooth quantitative samples. analyses. The Archimed Morphological® software features was used were in measured, order to record including digital stem images diameter, (Figure thickness 3) to ofrealize epidermis, qualitative primary and fibrequantitative layer, secondary analyses. fibres Morphological layer and xylem.features were measured, including stem diameter, thickness of epidermis, primary fibre layer, secondary fibres layer and xylem.

Fibers 2019, 7, 108 5 of 18 Fibers 2018, 6, x FOR PEER REVIEW 5 of 19

FigureFigure 3.3. Cross section of hemp stem (EL: epidermis layer, PFL: Primary fibresfibres layer, SFL:SFL: secondarysecondary fibresfibres layer).layer). 2.2.3. Biochemical Analysis 2.2.3. Biochemical Analysis The biochemical analyses were done using two methods described in details in our recent The biochemical analyses were done using two methods described in details in our recent work work [22], one based on solvent extractions to quantify cellulose, lignin and lipophilic extractives [22], one based on solvent extractions to quantify cellulose, lignin and lipophilic extractives contents contents according to several ASTM test methods (ASTM D 1107-56, ASTM D 1104-56, ASTM D according to several ASTM test methods (ASTM D 1107-56, ASTM D 1104-56, ASTM D 1103-60, 1103-60, ASTM D 1106-56 and ASTM D 1102-84), the other based on spectrophotometry to determine ASTM D 1106-56 and ASTM D 1102-84), the other based on spectrophotometry to determine the the pectins content. pectins content. 2.2.4. X-Ray Diffraction 2.2.4. X-Ray Diffraction The X-Ray diffraction (XDR) analyses were conducted on X-ray (XRD, AXS D8 Advance Bruker) The X-Ray diffraction (XDR) analyses were conducted on X-ray (XRD, AXS D8 Advance Bruker) diffractometer equipped with Cu-Kα radiation (λ = 1.54 Å). Measurements were performed on cut and diffractometer equipped with Cu-Kα radiation (λ = 1.54 Å). Measurements were performed on cut compressed fibres (disks of 25 mm in diameter and 2 mm in thickness). X-ray diffractograms were and compressed fibres (disks of 25 mm in diameter and 2 mm in thickness). X-ray diffractograms recorded within an angle range of 2θ from 5◦ to 70◦ with a scanning rate of 0.01 ◦/s. The crystalline were recorded within an angle range of 2θ from 5° to 70° with a scanning rate of 0.01 °/s. The order index (CI) was determined from X-ray diffractograms using a deconvolution method [22]. crystalline order index (CI) was determined from X-ray diffractograms using a deconvolution Individual peaks were extracted by a curve fitting process using Origin® software assuming Gaussian method [22]. Individual peaks were extracted by a curve fitting process using Origin® software functions. Crystalline peak areas (Ic) and amorphous broad peak area (Iam) were used to determinate assuming Gaussian functions. Crystalline peak areas (Ic) and amorphous broad peak area (Iam) the crystallinity index (CI) according to Equation (1). were used to determinate the crystallinity index (CI) according to Equation (1). Ic CICI == × 100 (1 (1)) Ic + Iam × 2.2.5. Thermogravimetric Analysis 2.2.5. Thermogravimetric Analysis Fibres collected at different harvest periods (BF, EF and SM) and retted at different times were analyzedFibres using collected a Perkin-Elmer at different Pyris-1 harvest thermal periods analysis (BF, EF system and SM) (PerkinElmer, and retted USA).at different Samples times of 8were mg wereanalyzed heated using in nitrogena Perkin- environmentElmer Pyris-1 from thermal 30 ◦ Canalysis to 700 system◦C at the (PerkinElmer, rate of 10 ◦C USA)./min. Samples At least twoof 8 samplesmg were of heated each batch in nitrogen were carried environment out. The from thermogravimetric 30 °C to 700 °C massat the loss rate (TG) of 10 and °C/min. mass lossAt least derivate two (DTG)samples were of recorded each batch as a were function carried of temperature. out. The thermogravimetric mass loss (TG) and mass loss derivate (DTG) were recorded as a function of temperature. 2.2.6. Tensile Test of Fibre Bundles 2.2.6. Tensile Test of Fibre Bundles The tensile characteristics of the fibres were measured using a micro-tensile testing instrument fromT DiaStronhe tensile Ltd. characteristics (Diastron Ltd., of the Hampshire, fibres were UK) measured [22]. Before using the a micro tensile-tensile tests, thetesting epidermis instrument was manuallyfrom DiaStron peeled Ltd. using (Diastron a razor Ltd., blade, Hampshire, and then UK) the fibre[22]. bundlesBefore the were tensile extracted tests, the carefully epidermis from was the stems.manually The peeled fibre bundlesusing a razor were blade, first glued and then on one-part the fibre plastic bundles tab were using extracted UV-curing carefully glue (DYMAX from the ultra-lightstems. The weld, fibre DYMAX bundles Europewere first GmbH, glued Wiesbaden, on one-part Germany). plastic tab Then, using the UV fibre-curing bundles glue diameters(DYMAX wereultra- determinedlight weld, DYMAX using an Europe automated GmbH, laser Wiesbaden, scanning deviceGermany). FDAS Then 765, the (Diastron fibre bundles Ltd., Hampshire, diameters UK).were Fibresdetermined horizontally using an rotated automated within laser the laserscanning beam device at 21 FDAS slices along765 (Diastron the fibre Ltd., axis. Hampshire, The mean, theUK). minimum Fibres horizontally and the maximum rotated within diameters the laser for beam each at slice 21 wereslices recordedalong the infibre a dimensionalaxis. The mean, report the minimum and the maximum diameters for each slice were recorded in a dimensional report exported by UvWin® 3.60 software. Thus, the mean of each slice along the fibre was averaged to calculate the effective cross-sectional area assuming that the fibre bundle had a circular shape. After Fibers 2019, 7, 108 6 of 18 exported by UvWin® 3.60 software. Thus, the mean of each slice along the fibre was averaged to calculate the effective cross-sectional area assuming that the fibre bundle had a circular shape. After that, the fibre bundles were automatically transported to the mini-tensile testing device (Diastron LEX810 system (Diastron Ltd., Hampshire, UK)) equipped with a 20 N capacity load cell. The samples were conditioned at a constant temperature (23 ◦C) and relative humidity (48%). The gauge length 1 was 12 mm and the crosshead displacement rate was 1.2 mm.min− . Approximately 30 specimens were tested for each treatment. The force to stress conversion was based on the aforementioned effective cross-sectional area. Young’s modulus (slope of linear zone of the curve), the tensile strength and the strain at break were determined using the obtained curve of force (Gram-Force) as function of displacement. The statistics analyses were carried out using ANalysis of VAriance (ANOVA) and Tukey multiple comparison test at a significant level of 5%.

3. Results and Discussion

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 (Figure4), 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 [25–27]. 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 [11,28]. The colour variation from yellow to grey for the fibres is related to the development of microbial communities (fungal and bacteria [29–31] 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 (Figure5) 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]. Fibers 2019, 7, 108 7 of 18 Fibers 2018, 6, x FOR PEER REVIEW 7 of 19 Fibers 2018, 6, x FOR PEER REVIEW 7 of 19

FigureFigure 4. 4. PhotographsPhotographs of of variation variation of of the the colo colourur during during field field retting retting of of stems stems and and fibres fibres harvested harvested at at differentdiFigurefferent 4. periods periodsPhotographs (BF, (BF, EF of and variation SM) SM).. of the colour during field retting of stems and fibres harvested at different periods (BF, EF and SM).

R0SM R1SM

100µm 100µm

100µm 100µm 1mm 1mm

1mm R3SM 1mm

R3SM

100µm

100µm 1mm

1mm

Figure 5. ESEM images of the surface change during field retting of the stems harvested at SM. Figure 5. ESEM images of the surface change during field field retting of the stems harvested at SM.SM. 3.2. Biochemical Composition 3.2. Biochemical Composition The evolution of relative biochemical composition as a function of retting duration is presented in FigureThe evolution6 for fibres ofof harvested relativerelative biochemicalbiochemical in the EF composition andcomposition SM period. as as a a functionThe function evolution of of retting retting for durationfibres duration harvested is is presented presented at BF in growthFigurein Figure6 stage for 6 fibresfor was fibres harvesteddetailed harvested in in a theprevious in EF the and EF paper SM and period. [22].SM period.The The results evolution The showevolution for that fibres thefor harvested fibresbiochemical harvested at BF variation growth at BF dependsstagegrowth was stage on detailed both was the detailed in harvest a previous in period a previous paper and [ 22the paper]. retting The [22]. results duration. The show results that show the biochemicalthat the biochemical variation variation depends ondepends bothFor theall on theharvestboth harvest the period harvest periods, and period the a grad retting andual the duration. increase retting duration.of cellulose content could be observed during field retting.For allall the Thetheharvest harvestcellulose periods, periods, increased a graduala gradfromual 63% increase increase to 77% of celluloseofafter cellulose 9 retting content content weeks could couldfor be fibres observed be observedharvested during during at fieldthe BFretting.field period retting. The [22], celluloseThe and cellulose from increased 68% increased to 75% from fromafter 63% 963% to retting 77% to 77% after weeks after 9 retting for 9 rettingfibres weeks harvested weeks for fibresfor atfibres EF harvested period, harvested atwhereas the at the BF itperiodBF increased period [22 ],[22], from and and from70% from to 68% 74% 68% to after 75%to 75% after5 retting after 9 retting 9 weeks retting weeks for weeks fibres for for fibres harvested fibres harvested harvested at SM at period. at EF EF period, period, In addition, whereas whereas an it increaseincreasedit increased of fromthe from cellulose 70% 70% to to 74%content 74% after after can 5 5 retting beretting noted weeks weeks from for for63% fibres fibres to 68% harvested harvested then to at at70% SM SM over period. period. the growth InIn addition,addition, period an fromincrease BF to of EF the and cellulose then to content SM, respectively. can be noted As from the quantification 63% to 68% then of cellulose to 70% content over the is growth relative, period thus, itsfrom variation BF to EF is andrelated then to to the SM, change respectively. of content As ofthe other quantification components. of cellulose The pectins content content is relative, decreased thus , its variation is related to the change of content of other components. The pectins content decreased Fibers 2019, 7, 108 8 of 18 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 ofFibers non-cellulosic 2018, 6, x FOR componentsPEER REVIEW (pectins and hemicelluloses). 9 of 19

Figure 6. Ash, lignin, lipophilic extractives, hemicelluloses, cellulose and lignin, pectins contents as aFigure function 6. Ash, of field lignin, retting lipophilic duration extractives, for fibres hemicelluloses, harvested at ( Acellulose) end of and flowering lignin, (EF)pectins and contents (B) at seed as a maturityfunction (SM).of field retting duration for fibres harvested at (A) end of flowering (EF) and (B) at seed maturity (SM). Figure6 shows also a decrease in minerals (ash) and lipophilic extractives (waxes, fats, resins) content3.3. Cellulose during Crystallinity 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 R0SMIn addition to 1.6% for to R5SM. the biochemical Lipophilic extractives analysis, the contents influence decreased of field from retting 14.9% onfor R0BF the cellulose to 4.1% 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), (101̅) 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, Fibers 2019, 7, 108 9 of 18 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 [29,30].

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 (Figure7). All the samples exhibit three defined peaks that are located at 2 θ diffraction angles of 14.8◦, 16.2◦ and 22.6◦, and which correspond to crystallographic planes of cellulose type I: (101), (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. Table2 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 [6,39–41]. Fibers 2018, 6, x FOR PEER REVIEW 10 of 19 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 celluloseFibers 2019 , structure,7, 108 as well as its degree of crystallization, may have a direct impact on10 the of 18 mechanical properties [6,39–41].

6000

5000 R5SM R3SM R9EF 4000 R5EF R3EF

3000 R0SM

R0EF

Relative intensity Relative 2000

1000

0 12 14 16 18 20 22 24 26 28 30 Diffraction angle 2degree FigureFigure 7. 7. XX-ray-ray diffractograms diffractograms of of the the fibres fibres collected collected at di atff differenterent retting retting duration duration for EF for and EF SM and periods. SM periods. Table 2. Evolution of crystallinity order index during retting of fibres harvested at EF and SM.

Table 2. Evolution of crystallinity order indexCrystallinity during retting Order of fibres Index harvested (%) at EF and SM. Retting Time EF SM Crystallinity Order R0 58Index (%) 64 R3 67 71 RettingR5 Time EF 68 SM 73 R9R0 58 69 64 - R3 67 71 3.4. Thermal Stability R5 68 73 R9 69 - 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 3.4. Thermal Stability stages. Figure8 shows the curves of TGA and DTGA obtained from di fferent retting durations of fibresA t harvestedhermogravimetric during the analysis EF and (TGA) SM periods. was carried Results out for in fibres order harvested to assess at the BF influence growth stage of field were rettingdetailed duration in a previous on the thermal paper [22 performance]. In general, of there the fibres are three harvested stages ofat decompositiondifferent hemp inplant TGA growth curves stages.(Figure Figure8A,C). 8 Theshows initial the weightcurves lossof TGA at about and 30–100DTGA obtained◦C is due from to the different evaporation retting of durations the absorbed of fibresmoisture. harvested The second during at the about EF and 230–260 SM period◦C is relateds. Results to the for decomposition fibres harvested of non-cellulosicat BF growth stage components were detailed(pectins in and a previous hemicelluloses) paper [22]. and In the general, third stage there at are 335 three◦C is stages attributed of decompos to the decompositionition in TGA ofcu majorrves (Figurecomponent 8A,C). of The fibres initial (cellulose). weight loss This at process about 30 of– decomposition100 °C is due to of the the evaporation fibres is consistent of the absorbed with data moreportedisture. in The literature second [12 at, 22 about,42–44 230]. –260 °C is related to the decomposition of non-cellulosic componentsIn TGA (pectins curves, peaksand are hemicelluloses) superimposed and on athe temperature third stage scale, at thus, 335 by °C calculating is attributed the derivative to the decompositionof the weight lossof major as a function component of temperature of fibres (cellulose). (Figure8 B,D).This Theprocess decomposition of decomposition process of of the the fibres fibres iscan consistent be clearly with observed data reported in this in case. literature From [12,22,42 the peak–44 with]. the highest intensity, it can be seen that theIn increase TGA in curves, the retting peaks duration are superimposed led to an increase on a oftemperature the decomposition scale, thus temperature, by calculating of the fibres, the derivativefrom 337 ◦ofC the to 359weight◦C for loss R0BF as a andfunction R9BF, of from temperature 323 ◦C to (Figure 357 ◦C for8B, R0EFD). The and decomposition R9EF and from process 350 ◦C of the fibres can be clearly observed in this case. From the peak with the highest intensity, it can be to 367 ◦C for R0SM and R5SM. This emphasized a higher thermal stability for high retted fibres, as confirmed by the literature [12,44,45]. 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 Fibers 2018, 6, x FOR PEER REVIEW 11 of 19 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 [12,44,45]. 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 Fibers 2019, 7, 108 11 of 18 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 phenomenonstructural order brings of cellulose a higher with structural strong order intramolecular of cellulose and with molecular strong intramolecular hydrogen bonds and that molecular need a hydrogenhigher degradation bonds that temperature need a higher to bedegradation broken down temperature [43,44]. to be broken down [43,44].

(B) (A) 12 100 Cellulose 90 10 R0EF 80 R3EF Cellulose R5EF R0EF 70 8 R9EF R3EF 60 R5EF R9EF 6 50

Weight (%) Weight 40 4

30 %/min) ( derivative Weight

20 2

10 0 0 30 130 230 330 430 530 630 30 130 230 330 430 530 630 Temperature (°C) Temperature (°C) (D) (C) 12 100 Cellulose 90 10 R0SM 80 R3SM R5SM Cellulose 70 8 R0SM 60 R3SM R5SM 6

50 Weight (%) Weight 40 4

30 %/min) ( derivative Weight

20 2

10 0 0 30 130 230 330 430 530 630 30 130 230 330 430 530 630 Temperature (°C) Temperature (°C) FigureFigure 8. TGATGA ( (AA)) and and DTGA DTGA ( (BB)) curves curves of of fibres fibres harvested harvested at at EF EF (R0EF) (R0EF) and and retted retted at at different different times times (R3EF,(R3EF, R5EF R5EF and and R9EF). R9EF). TGA TGA (C ) (C and) and DTGA DTGA (D) of (D fibres) of fibres harvested harvested at SM at(R0SM) SM (R0SM) and retted and at retted different at differenttimes (R3SM, times R5SM). (R3SM, R5SM).

3.5. Morphology Morphology TheThe evolution evolution in in plant plant morphology morphology of ofhemp hemp during during three three growth growth stages stages (BF, (BF,EF and EF SM) and was SM) investigated.was investigated. Hemp Hemp stems containedstems contained different di ff layerserent, layers,as already as already described described by number by number of authors of [13,46,47authors [].13 They,46,47 are]. They organized are organized from the from stem the pith stem toward pith the toward surface the by surface central by woody central core woody (xylem core designed(xylem designed as X), cambium, as X), cambium, cortex (inc cortexluding (including both primary both primary-and secondary -and secondary fibres noted fibres PFB noted and PFBSFB respectively)and SFB respectively) and epidermis and epidermis (called E). (called During E). During growth growth from the from beginning the beginning of flowering of flowering to the 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 (Figure9). 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. Fibers 2018, 6, x FOR PEER REVIEW 12 of 19 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 Fibers 2019, 7, 108 12 of 18 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 qualitativelyThe impact analyzed. of field retting Figure duration 10 shows on the the optical morphology microscope of the hemp micrographs fibres was of then cross qualitatively-sections of analyzed.unretted (R0EF) Figure and10 shows retted the hemp optical stems microscope (R3EF, R5EF micrographs and R9EF) of cross-sections collected at ofthe unretted end of (R0EF)flowering. and rettedWhen hempthe hemp stems stem (R3EF, is unretted R5EF and (R0EF), R9EF) the collected structure at the of enddifferent of flowering. layers of When the stem the hempis intact stem and is unrettedwell organized. (R0EF), The the structurefibres are of gathered different layersin the ofform the stemof a bundle is intact and and the well lumen organized. of the The elementary fibres are gatheredfibres cannot in the be form distinctly of a bundle observed. and Since the lumen that field of the retting elementary period fibres of stems cannot harvested be distinctly at EF observed.was long Sinceand slow, that fieldno high retting difference period ofcould stems be harvested observed atafter EF was3 weeks long and(R3EF) slow, of noretting high at di fftheerence level could of bast be observedfibre. In contrast, after 3 weeks after (R3EF)5 weeks of of retting retting at (R5EF), the level the of baststructure fibre. of In stem contrast, changed. after 5The weeks primary of retting and (R5EF),secondary the fibre structure bundles of stem were changed. separated The into primary smaller and fibres secondary bundles fibre, resulting bundles in were more separated open spaces into smallerbetween fibres the fibre bundles, bundles. resulting When inthe more stems open were spaces highly between retted (R9EF), the fibre the bundles. structure Whenof the thestem stems was wereaffected highly by fieldretted retting. (R9EF), The the structureepidermis of layer the stem was wasdeformed affected and by field removed. retting. The The bundles epidermis of fibres layer waswere deformed completely and separated removed. into The bundleselementary of fibres fibres were (EF) completely and their separated lumen (L) into is clearly elementary visible. fibres A (EF)similar and evolution their lumen of the (L) morphology is clearly visible. of the Ahemp similar fibres evolution during ofthe the field morphology retting process of the was hemp observed fibres duringin our recent the field work retting [22] process for stems was harvested observed inat our BF. recent Overall, work whatever [22] for stemsthe growth harvested period, at BF. the Overall, same whateverevolution theof the growth morphology period, of the the same hemp evolution fibres during of the morphologyfield retting process of the hemp is highlighted. fibres during The fieldbast rettingfibre bundles process are is highlighted. separated ( Thei) from bast thefibre central bundles woody are separated core and (i) epidermisfrom the central (ii) and woody into core smaller and epidermisbundles or (ii)individual and into fibres. smaller bundles or individual fibres. This change in morphology during retting is due to the microorganisms’microorganisms’ activities that would allow the the removal removal of intercellular of intercellular cementing cementing components components (pectins (pectins and lipids and extractives) lipids extractives) in agreement in withagreement the biochemical with the analyses. biochemical This analyses. separation This of the separation fibres into of smaller the fibres bundles into or smaller elementary bundles fibres or duringelementary field fibres retting during would field have retting a positive would ehaveffect ona positive the mechanical effect on propertiesthe mechanical of hemp properties fibres asof reinforcementshemp fibres as reinforcements in composites [ 11in ,composites16,17]. [11,16,17].

R0BF Bast fibres

R0EF Xylem

Central void

R0SM

0 500 1000 1500 2000 2500 3000 3500 Thickness (µm)

0 30 60 90 120 150 180

R0BF EL

R0EF PFL

SFL R0SM

Figure 9. Morphological features (bast fibres,fibres, xylem, and central void). EL: EL: Epidermis Epidermis layer, layer, PFL: primary fibresfibres layer, layer, SFL: SFL: secondary secondary fibres fibres layer layer of hemp of hemp stem stem depending depending on harvest on harvest period period before retting.before retting. Fibers 2019, 7, 108 13 of 18 Fibers 2018, 6, x FOR PEER REVIEW 13 of 19

R0EF R3EF 100µ m 100µ m

X X

R5EF R9EF 100µ m 100µ m

X X

Figure 10. Optical microscope micrographs of cross sections of R0EF, R3EF, R5EF, R9EF; E: epidermis; Figure 10. Optical microscope micrographs of cross sections of R0EF, R3EF, R5EF, R9EF; E: epidermis; PFB: primary fibre bundles; SFB: secondary fibre bundles; ML: middle lamella; L: lumen; EF: elementary PFB: primary fibre bundles; SFB: secondary fibre bundles; ML: middle lamella; L: lumen; EF: fibres; X: xylem. elementary fibres; X: xylem. 3.6. Tensile Properties 3.6. Tensile Properties In order to evaluate the influence of field retting on the hemp fibres harvested at different growthIn orderstages to (BF, evaluate EF and theSM), influence the mechanical of field properties retting on of the the fibrehemp bundles fibres harveste were characterizedd at different by growthmicro-tensile stages tests. (BF, EF As and shown SM), in the Figure mechanical 11A, the properties fibre bundle of the diameters fibre bundles selected were for characterized each batch were by microapproximately-tensile tests. between As shown 100 and in Figure 240 µm 11A, with the a median fibre bundle value diameters at about 160 selectedµm so for that each it was batch possible were approximatelyto compare relatively between the 100 results and 240 of micro-tensile µm with a median tests. Figurevalue at 11 aboutA–D presents160 µm so the that tensile it was properties possible (tensileto compare strength, relatively Young’s the modulusresults of andmicro strain-tensile at failure) tests. Figure of the fibre11A– bundlesD present extracteds the tensile at the properties different (tensilegrowth stages strength, and Young’s retted at dimodulusfferent times. and strain at failure) of the fibre bundles extracted at the differentAs concerns growth stages the influence and retted of theat different growth stage,times. an increase of all the mechanical characteristics (consideringAs concerns median the values)influence with of plantthe growth growth stage, can be an observed. increase Indeedof all the the mechanical tensile strength characteristics increased (consideringfrom 174 MPa median for the R0BF values) to 331 with MPa plant for growth R0EF and can then be reached observed up. toIndeed 352 MPa the for tensile R0SM. strength Fibres increasharvesteded from at BF 174 (R0BF) MPa have for the a lower R0BF tensile to 331 strength MPa for than R0EF that and of then the fibres reached harvested up to 352 during MPa the for EF R0SM. and FibresSM periods, harvested while at noBF significant (R0BF) have diff aerence lower (tensilep > 0.05) strength is observed than that between of the R0EF fibres and harvested R0SM. Young’s during themodulus EF and increased SM period significantlys, while from no significant 8 GPa for differenceR0BF to 13 ( GPap > 0.05) for R0EF is observed and R0SM. between A similar R0EF trend and is R0SM.observed Young’s for strain modulus at break. increased It increased significantly significantly from from8 GPa 2.4% for toR0BF 3.2% to and13 GPa 3.4% for for R0EF fibres and harvested R0SM. Aduring similar the trend BF, EF is andobserved SM periods, for strain respectively. at break. AccordingIt increased to significantly these results, from it can 2.4% be concluded to 3.2% and that 3.4% the formain fibres improvement harvested ofdu tensilering the properties BF, EF and occurred SM period betweens, respectively. the fibres According extracted at to the these end results, of flowering it can (R0EF)be concluded and the that beginning the main of improvement flowering (R0BF), of tensile as just properties a slight increase occurred was between observed the for fibres fibres extracted collected at the seedend of maturity flowering period (R0EF) (R0SM). and the This beginning change in of tensile flowering properties (R0BF), during as jus plantt a slight growth increase might was be observeddue to the for variation fibres collected in the biochemical at the seed composition, maturity period fibres (R0SM). morphology This andchange fibres in extraction.tensile properties Indeed, duringthe increase plant of growth cellulose might fraction be with due growth to the stage variation could in play the a keybiochemical role in increasing composition, mechanical fibres morphologyperformance and [6,23 fibres]. Moreover, extraction. Goudenhooft Indeed, the et increase al. [10] highlightedof cellulose thatfraction the morphologywith growth ofstage the could fibres playcould a also key impact role in the increasing mechanical mechanical performance. performance They reported [6,23 an]. increase Moreover, of the Goudenhooft mechanical etproperties al. [10] highlightedof flax fibres that during the plantmorphology development. of the Anotherfibres could explanation also impact could the be mechanical attributed to performance. the fibres’ damage 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 Fibers 2019, 7, 108 14 of 18 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 [39–41]. 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 Fibers 2019, 7, 108 15 of 18 properties of the initial state of the fibres after harvesting were high (ii) the retting period coincides withFibers two 2018 seasons, 6, x FOR (summer PEER REVIEW and autumn) which allows the mastering of retting mechanisms.15 of 19

B 1000 BF EF SM

800

600

400

200

Tensile strength (MPa)

R0BF R3BF R5BF R9BF R0EF R3EF R5EF R9EF R0SM R1SM R3SM R5SM

Retting duration (weeks)

C 40 BF EF SM

Young's modulus (GPa) 0

R0BF R3BF R5BF R9BF R0EF R3EF R5EF R9EF R0SM R1SM R3SM R5SM

Retting duration (weeks)

D 8 BF EF SM

2 Strain Strain at break (%)

Elongation at break (%)

R0BF R3BF R5BF R9BF R0EF R3EF R5EF R9EF R0SM R1SM R3SM R5SM

Retting duration (weeks)

FigureFigure 11. 11.Scattegram Scattegram of ofmeasured measured bundlebundle fibres ﬁbres diameters for for each each batch batch (A (A); );Box Box plots plots of oftensile tensile strengthstrength (B ),(B Young’s), Young’s modulus modulus (C (),C), and and stain stain at at break break (D (D) for) for unretted unretted and and retted retted ﬁbres fibres harvested harvested atat BF, EFBF, and EF SM. and The SM. back The crossesback crosses and redand linered correspondline correspond to the to the means means and and medians, medians, respectively. respectively.

Fibers 2019, 7, 108 16 of 18

4. Conclusions A comparison of the effect of field retting duration on the properties of the hemp fibres harvested at different growth stages (beginning of flowering, end of flowering, and seed maturity) was examined in this work. The retting of these periods was performed under different weather conditions. The fibres harvested at BF and EF showed a similar evolution in colour change from light green for unretted samples (R0), yellow for low retted samples (R1 to R3), yellow with the presence of grey fibres for medium retted samples (R4 and R5) and grey for highly retted samples (R9). However, for the seed maturity (SM) period, the colour transition from yellow to dark grey was rapid (3 weeks). This colour change is related to weather conditions and the development of microorganisms at surface stem (ESEM images). TGA, biochemical and XRD measurements showed an increase of cellulose fraction and its degree of crystallinity during retting for all the examined harvest periods. Nonetheless, the kinetics of this variation were not identical during retting of each examined harvest periods, since each harvest period was field retted with specific climatic conditions. Furthermore, during plant development, the cellulose fraction was increased (e.g., from 68% for R0EF to 70% for R0SM) with the removal of non-cellulosic materials. The increase of cellulose during retting led to a progressive improvement of thermal stability. The fibres harvested at SM exhibit a better thermal stability compared to that of the fibres harvested at BF and EF, due to the high cellulose fraction present in the fibres at plant maturity. The change in the biochemical composition, especially the degradation of cementing components (e.g., pectins and lipids) during retting led to the removal of epidermal and parenchyma cells, resulting in the separation of bundle of fibres to individual elementary fibres. The tensile properties increased during the retting of fibres harvested at BF and EF to a maximum value (after 5 weeks) and decreased at the end of retting. As concern the fibres harvested at SM, the stems started to be slightly retted before even being harvested, thus, under the very rainy retting period, the tensile properties decreased progressively and slightly. The increase of the cellulose fraction and the cellulose chain packing order allows better tensile performances of the fibres before reaching over-retting. When non-cellulosic materials are highly degraded, the coherence between the cellulose microfibrils and non-cellulosic matrix becomes weaker and thereby, results in lower tensile properties. Then, in order to avoid an under- and over-retting treatment, it seems to be judicious to control both criteria: initial intrinsic characteristics of the fibres at the harvesting period and during the retting process as retting kinetics are closely linked with climatic conditions. From these data, reliable tools could be provided for farmers in order to better manage the retting process. The improvement of dew-retting management could lead to the development of composite material with a highly reproducible performance.

Author Contributions: A.B., J.-C.B. and L.M. and designed the research. B.M. performed the research, analyzed the data and wrote the manuscript. All authors read the manuscript and approved it. Funding: This research received no external funding Acknowledgments: The authors are grateful to the CIVAM Chanvre Gardois association (Bouquet, France) for their collaboration to conduct this study in their field. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Duval, A.; Bourmaud, A.; Augier, L.; Baley, C. Influence of the sampling area of the stem on the mechanical properties of hemp fibers. Mater. Let. 2011, 65, 797–800. [CrossRef] 2. Bourmaud, A.; Beaugrand, J.; Shah, D.U.; Placet, V.; Baley, C. Towards the design of high-performance plant fibre composites: How can we best define the diversity and specificities of plant cell walls? Prog. Mater. Sci. 2018, 97, 347–408. [CrossRef] 3. Islam, M.S.; Pickering, K.L.; Foreman, N.J. Influence of alkali fiber treatment and fiber processing on the mechanical properties of hemp/epoxy composites. J. Appl. Polym. Sci. 2010, 21, 449–456. [CrossRef] Fibers 2019, 7, 108 17 of 18

4. Stamboulis, A.; Baillie, C.A.; Peijs, T. Effects of environmental conditions on mechanical and physical proprties of flax fibers. Compos. Part A 2001, 32, 1105–1115. [CrossRef] 5. Aziz, S.H.; Ansell, M.P. The effect of alkalization and fibre alignment on the mechanical and thermal properties of kenaf and hemp bast fibre composites: Part 1-polyester resin matrix. Compos. Sci. Technol. 2004, 64, 1219–1230. [CrossRef] 6. Mazian, B.; Bergeret, A.; Benezet, J.-C.; Malhautier, L. Impact of field retting and accelerated retting performed in a lab-scale pilot unit on the properties of hemp fibres/polypropylene biocomposites. Ind. Crops Prod. 2020, 143, 111912. [CrossRef] 7. Marrot, L.; Lefeuvre, A.; Pontoire, B.; Bourmaud, A.; Baley, C. Analysis of the hemp fiber mechanical properties and their scattering (Fedora 17). Ind. Crops Prod. 2013, 51, 317–327. [CrossRef] 8. Van der Werf, H.M.G.; Wijlhuizen, M.; de Schutter, J.A.A. Plant density and self-thinning affect yield and quality of fibre hemp (Cannabis sativa L.). Fields Crops Res. 1995, 40, 153–164. [CrossRef] 9. Mediavilla, V.; Leupin, M.; Keller, A. Influence of the growth stage of industrial hemp on the yield formation in relation to certain fibre quality traits. Ind. Crops Prod. 2001, 13, 49–56. [CrossRef] 10. Goudenhooft, C.; Siniscalco, D.; Arnould, O.; Bourmaud, A.; Sire, O.; Gorshkova, T.; Baley, C. Investigation of the mechanical properties of flax cell walls during plant development: The relation between performance and cell wall structure. Fibers 2018, 6, 6. [CrossRef] 11. Martin, N.; Mouret, N.; Davies, P.; Baley, C. Influence of the degree of retting of flax fibers on the tensile properties of single fibers and short fiber/polypropylene composites. Ind. Crops Prod. 2013, 49, 755–767. [CrossRef] 12. Placet, V.; Day, A.; Beaugrand, J. The influence of unintended field retting on the physicochemical and mechanical properties of industrial hemp bast fibres. J. Mater. Sci. 2017, 52, 5759–5777. [CrossRef] 13. Liu, M.; Fernando, D.; Daniel, G.; Madsen, B.; Meyer, A.S.; Ale, M.T.; Thygesen, A. Effect of harvest time and field retting duration on the chemical composition, morphology and mechanical properties of hemp fibers. Ind. Crops Prod. 2015, 69, 29–39. [CrossRef] 14. Hearle, J.W. The fine structure of fibers and crystalline. J. Appl. Polym. Sci. 1963, 7, 1175–1192. [CrossRef] 15. Crônier, D.; Monties, B.; Chabbert, B.R. Structure and chemical composition of bast fibers isolated from developing hemp stem. J. Agric. Food Chem. 2005, 53, 8279–8289. [CrossRef] 16. Li, Y.; Pickering, K.L.; Farrell, R.L. Analysis of green hemp fibre reinforced composites using bag retting and white rot fungal treatments. Ind. Crops Prod. 2009, 29, 420–426. [CrossRef] 17. Sisti, L.; Totaro, G.; Vannini, M.; Fabbri, P.; Kalia, S.; Zatta, A.; Celli, A. Evaluation of the retting process as a pre-treatment of vegetable fibers for the preparation of high-performance polymer biocomposites. Ind. Crops Prod. 2016, 81, 56–65. [CrossRef] 18. Paridah, M.T.; Basher, A.B.; SaifulAzry, S.; Ahmed, Z. Retting process of some bast plant fibres and its effect on fibre quality: A review. BioResources 2011, 6, 5260–5281. 19. Bacci, L.; Di Lonardo, S.; Albanese, L.; Mastromei, G.; Perito, B. Effect of different extraction methods on fiber quality of nettle (Urtica dioica L.). Text. Res. J. 2011, 81, 827–837. [CrossRef] 20. Akin, D.E.; Condon, B.; Sohn, M.; Foulk, J.A.; Dodd, R.B.; Rigsby, L.L. Optimization for enzyme-retting of flax with pectate lyase. Ind. Crops Prod. 2007, 25, 136–146. [CrossRef] 21. Henriksson, G.; Akin, D.E.; Hanlin, R.T.; Rodriguez, C.; Archibald, D.D.; Rigsby, L.L.; Eriksson, K.L. Identification and retting efficiencies of fungi isolated from dew-retted flax in the United States and europe. Appl. Environ. Microbiol. 1997, 63, 3950–3956. 22. Mazian, B.; Bergeret, A.; Benezet, J.-C.; Malhautier, L. Influence of field retting duration on the biochemical, microstructural, thermal and mechanical properties of hemp fibres harvested at the beginning of flowering. Ind. Crops Prod. 2018, 116, 170–181. [CrossRef] 23. Charlet, K.; Baley, C.; Morvan, C.; Jernot, J.P.; Gomina, M.; Bréard, J. Characteristics of Hermès flax fibres as a function of their location in the stem and properties of the derived unidirectional composites. Compos. Part A Appl. Sci. Manuf. 2007, 38, 1912–1921. [CrossRef] 24. Liu,M.; Fernando,D.; Meyer,A.S.; Madsen,B.; Daniel,G.; Thygesen,A.Characterizationandbiologicaldepectinization of hemp fibers originating from different stem sections. Ind. Crops Prod. 2015, 76, 880–891. [CrossRef] 25. Hörtensteiner, S. The loss of green color during chlorophyll degradation—A prerequisite to prevent cell death? Planta 2004, 219, 191–194. [CrossRef] 26. Merzlyak, M.N.; Gitelson, A. Why and what for the leaves are yellow in autumn? On the interpretation of optical spectra of senescing leaves (Acerplatanoides L.). J. Plant Physiol. 1995, 145, 315–320. [CrossRef] Fibers 2019, 7, 108 18 of 18

27. Matile, P. Biochemistry of indian summer: Physiology of autumnal leaf coloration. Exp. Gerontol. 2000, 35, 145–158. [CrossRef] 28. Pallesen, B.E. The quality of combine-harvested fibre flax for industrials purposes depends on the degree of retting. Ind. Crops Prod. 1996, 5, 65–78. [CrossRef] 29. Ribeiro, A.; Pochart, P.; Day, A.; Mennuni, S.; Bono, P.; Baret, J.; Spadoni, J.; Mangin, I. Microbial diversity observed during hemp retting. Appl. Microbiol. Biotechnol. 2015, 99, 4471–4484. [CrossRef] 30. Liu, M.; Ale, M.T.; Kołaczkowski, B.; Fernando, D.; Daniel, G.; Meyer, A.S.; Thygesen, A. Comparison of traditional field retting and Phlebia radiata Cel 26 retting of hemp fibres for fibre-reinforced composites. AMB Express 2017, 7, 58. [CrossRef] 31. Bleuze, L.; Lashermes, G.; Alavoine, G.; Recous, S.; Chabbert, B. Tracking the dynamics of hemp dew retting under controlled environmental conditions. Ind. Crops Prod. 2018, 123, 55–63. [CrossRef] 32. Nykter, M.; Kyma, H.; Belinda, A.; Lilholt, H.; Thygesen, A. Effects of thermal and enzymatic treatments and harvesting time on the microbial quality and chemical composition of fibre hemp (Cannabis sativa L.). Biomass Bioenergy. 2008, 32, 392–399. [CrossRef] 33. Meijer, W.J.M.; Vertregt, N.; Rutgers, B.; van de Waart, M. The pectin content as a measure of the retting and rettability of flax. Ind. Crops Prod. 1995, 4, 273–284. [CrossRef] 34. Musialak, M.; Wróbel-Kwiatkowska, M.; Kulma, A.; Starzycka, E.; Szopa, J. Improving retting of fibre through genetic modification of flax to express pectinases. Transgenic Res. 2008, 17, 133–147. [CrossRef] 35. Foulk, J.A.; Akin, D.E.; Dodd, R.B. Processing techniques for improving enzyme-retting of flax. Ind. Crops Prod. 2001, 13, 239–248. [CrossRef] 36. Tserki, V.; Zafeiropoulos, N.E.; Simon, F.; Panayiotou, C. A study of the effect of acetylation and propionylation surface treatments on natural fibres. Compos. Part A Appl. Sci. Manuf. 2005, 36, 1110–1118. [CrossRef] 37. Li, Y.; Pickering, K.L.; Farrell, R.L. Determination of interfacial shear strength of white rot fungi treated hemp fibre reinforced polypropylene. Compos. Sci. Technol. 2009, 69, 1165–1171. [CrossRef] 38. Zafeiropoulos, N.E.; Baillie, C.A.; Matthews, F.L. Study of transcrystallinity and its effect on the interface in flax fibre reinforced composite materials. Compos. Part A Appl. Sci. Manuf. 2001, 32, 525–543. [CrossRef] 39. Sawpan, M.A.; Pickering, K.L.; Fernyhough, A. Effect of various chemical treatments on the fibre structure and tensile properties of industrial hemp fibres. Compos. Part A Appl. Sci. Manuf. 2011, 42, 888–895. [CrossRef] 40. Bledzki, A.; Gassan, J. Composites reinforced with cellulose based fibres. Prog. Polym. Sci. 1999, 24, 221–274. [CrossRef] 41. Placet, V.; Trivaudey, F.; Cisse, O.; Gucheret-Retel, V.; Boubakar, M.L. Diameter dependence of the apparent tensile modulus of hemp fibres: A morphological, structural or ultrastructural effect? Compos. Part A Appl. Sci. Manuf. 2012, 43, 275–287. [CrossRef] 42. Ghetti, P.; Ricca, L.; Angelini, L. Thermal analysis of biomass and corresponding pyrolysis products. Fuel 1996, 75, 565–573. [CrossRef] 43. Ouajai, S.; Shanks, R.A. Composition, structure and thermal degradation of hemp cellulose after chemical treatments. Polym. Degrad. Stab. 2005, 89, 327–335. [CrossRef] 44. Kabir, M.M.; Wang, H.; Lau, K.T.; Cardona, F. Effects of chemical treatments on hemp fibre structure. Appl. Surf. Sci. 2013, 276, 13–23. [CrossRef] 45. Manikandan Nair, K.C.; Thomas, S.; Groeninckx, G. Thermal and dynamic mechanical analysis of polystyrene composites reinforced with short sisal fibres. Compos. Sci. Technol. 2001, 61, 2519–2529. [CrossRef] 46. Tanja Schäfer, B.H. Effect of sowing date and plant density on the cell morphology of hemp (Cannabis sativa L.). Ind. Crops Prod. 2006, 23, 88–98. 47. Réquilé, S.; Goudenhooft, C.; Bourmaud, A.; Le Duigou, A.; Baley, C. Exploring the link between flexural behaviour of hemp and flax stems and fibre stiffness. Ind. Crops Prod. 2018, 113, 179–186. [CrossRef] 48. Bourmaud, A.; Morvan, C.; Bouali, A.; Placet, V.; Perré, P.; Baley, C. Relationships between micro-fibrillar angle, mechanical properties and biochemical composition of flax fibers. Ind. Crops Prod. 2013, 44, 343–351. [CrossRef] 49. Placet, V.; François, C.; Day, A.; Beaugrand, J.; Ouagne, P. Industrial hemp transformation for composite applications: Influence of processing parameters on the fibre properties. Adv. Nat. Fibre Compos. 2018, 13–25.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).