Elucidating field retting mechanisms of hemp fibres for biocomposites: Effects of microbial actions and interactions on the cellular micro-morphology and ultrastructure of hemp stems and bast fibres

Abstract

Field retting is an industrial process for extracting valuable bast fibres from hemp. In this study, molecular, chemical, and scanning electron microscopy studies were employed to understand the field retting mechanisms involving microbiota, including microbial community dynamics, hemp colonization, functions/interactions, and hemp biodegradation. This study for the first time revealed the coexistence of bacterial-fungal interactions during retting and showed progressive microbial breakdown of the stems. Using scanning electron microscopy, evidence for microbial activities/interactions within the stems was obtained, which helped to understand hemp retting mechanisms. The collective findings showed that: a) initially, easily accessible food within the hemp stems attracted and supported microbial invasion and decay, with activities influenced by the stem anatomy, chemistry, and morphology; b) filamentous fungi as key players in the early stages remarkably contributed to efficient fibre defibration; c) extended retting enhanced the bacterial activities, including bacterial-fungal interactions and their dominant role within the community; d) bacterial attraction and activities were promoted by bacterial mycophagy with a set of different phenotypic behaviours for nutrients and fungal highways for transport within the stems; and e) bast fibre degradation leading to inferior quality during prolonged retting was caused by ultrastructural modifications to all of the major fibre cell wall layers.

Full Article

Elucidating Field Retting Mechanisms of Hemp Fibres for Biocomposites: Effects of Microbial Actions and Interactions on the Cellular Micro-morphology and Ultrastructure of Hemp Stems and Bast Fibres

Dinesh Fernando,^a,* Anders Thygesen,^b Anne S. Meyer,^b and Geoffrey Daniel ^a

Field retting is an industrial process for extracting valuable bast fibres from hemp. In this study, molecular, chemical, and scanning electron microscopy studies were employed to understand the field retting mechanisms involving microbiota, including microbial community dynamics, hemp colonization, functions/interactions, and hemp biodegradation. This study for the first time revealed the coexistence of bacterial-fungal interactions during retting and showed progressive microbial breakdown of the stems. Using scanning electron microscopy, evidence for microbial activities/interactions within the stems was obtained, which helped to understand hemp retting mechanisms. The collective findings showed that: a) initially, easily accessible food within the hemp stems attracted and supported microbial invasion and decay, with activities influenced by the stem anatomy, chemistry, and morphology; b) filamentous fungi as key players in the early stages remarkably contributed to efficient fibre defibration; c) extended retting enhanced the bacterial activities, including bacterial-fungal interactions and their dominant role within the community; d) bacterial attraction and activities were promoted by bacterial mycophagy with a set of different phenotypic behaviours for nutrients and fungal highways for transport within the stems; and e) bast fibre degradation leading to inferior quality during prolonged retting was caused by ultrastructural modifications to all of the major fibre cell wall layers.

Keywords: Bacterial-fungal interactions (BFIs); Bacterial mycophagy; Fibre cell wall ultrastructure; Field retting; Hemp fibres; Microbial dynamics; Scanning electron microscopy (SEM)

Contact information: a: Department of Forest Biomaterials and Technology, Swedish University of Agricultural Science (SLU), P. O. Box 7008, SE-750 07 Uppsala, Sweden; b: Protein Chemistry & Enzyme Technology, DTU Bioengineering, Building 221, Technical University of Denmark, 2800 Kongens Lyngby, Denmark; *Corresponding author: dinesh.fernando@slu.se

INTRODUCTION

Hemp (Cannabis sativa L.) is a multi-purpose annual plant that produces natural fibres (i.e., bast fibres) and has gained increasing interest because of the industrial potential of the plant in the construction, pulp and paper, and textiles industries, and as a reinforcement material to replace synthetic fibre-reinforced plastics (Corbière-Nicollier et al. 2001; Pickering et al. 2007; Amaducci and Gusovius 2010; Andre et al. 2016; Liu et al. 2017b). Hemp is also a renewable fibre resource that provides integrated ecological (i.e., renewability, low emission of toxic fumes, biodegradability, and biocompatibility) and economic benefits (Corbière-Nicollier et al. 2001; Joshi et al. 2004; Tahir et al. 2011). Hemp provides composite manufacturers with several advantages, including a low cost, high mechanical properties (stiffness and strength) combined with a low density, flexibility, and reduced tool wear during processing (Fan et al. 2011; Tahir et al. 2011; Liu et al. 2016; Pickering et al. 2016).

Hemp bast fibres (also generally referred as “fibres”) are cellulose-rich sclerenchymatous cells comprised of both primary and secondary fibres that are located in the cortex below the outer epidermis, which forms a multi-layered ring that encircles the core xylem. In hemp stems, bast fibres are characterized by thick secondary cell walls, whose chemical and morphological structures are distinct from those of the core xylem fibres. Hemp fibre cell walls are natural composites similar to wood fibres and their main components are highly crystalline cellulose, hemicellulose, lignin, and pectin (Crônier et al. 2005; Wang et al. 2007; Liu et al. 2015). Bast fibres are intimately associated forming fibre bundles (i.e., several fibre cells grouped together as clusters) via pectin and lignin-rich middle lamellae (ML). Individual bundles are held together by delicate living parenchyma cells, whose cell walls are composed primarily of pectic substances (Liu et al. 2015).

One major step for the industrial use of bast fibres (e.g., for biocomposites) is improved and efficient fibre processing (i.e., fibre extraction), where the main principle is to remove the cementing materials between the fibres and fibre bundles to obtain individual cellulose-rich fibres with minimum damage. The isolation of cellulosic fibres from non-fibrous tissues and woody parts of hemp stems (i.e., retting) poses a major challenge that has considerable bearing on the subsequent chemical and physical properties of single fibres. In particular, damage leads to a greater variability in the mechanical performance of fibres as reinforcing agents, which renders them unsuitable for use in high-grade composite materials (Easson and Molly 1996; Liu et al. 2015; Pickering et al. 2016). For example, manually extracted flax fibres have been shown to have a 20% higher strength than those extracted mechanically (Bos et al.2002). Traditionally, bast fibres are extracted using either field (dew) or water retting (Di Candilo et al. 2010; Jankauskienė et al. 2015). The impact on the quality of the extracted fibres largely depends on environmental factors, like the agro-climatic conditions (i.e., rainfall and temperature), underlying soil, and most importantly the retting duration (Tahir et al. 2011; Das et al. 2014; Liu et al. 2015; Ribeiro et al. 2015).

Field retting is basically a biochemical process, where partial decomposition of plant stems occurs under native conditions. This facilitates the separation of bast fibres from hemp stems during subsequent mechanical processing (decortication) and represents a key process for obtaining high-quality raw materials (Easson and Molly 1996; Tahir et al. 2011; Das et al. 2014). The main purpose is to degrade the binding materials (i.e., pectin) between the individual fibres and fibre bundles, and other cementing compounds (hemicellulose and lignin) in the ML using a pool of microbiota (Akin 2013; Ribeiro et al. 2015). Previously, fungi were considered the major active microflora responsible for dew retting (Fuller and Norman 1944). However, recent studies have shown a complex microbial community with both bacteria (e.g., Pseudomonas and Escherichia coli) and fungi (Cryptococcus and Cladosporium) causing enzymatic hydrolysis of pectin via pectinases (i.e., polygalacturonase, pectin lyases, and pectin esterases) (Alkorta et al. 1998; Munshi and Chatto 2008; Ribeiro et al. 2015). However, natural retting is difficult to control and has inherent drawbacks, such as inferior fibre properties from over retting, heterogeneity in the mechanical properties of the extracted fibres, and negative environmental impacts from water pollution (Hu et al. 2012). Because of these concerns and costs related to labour and drying during anaerobic water retting, aerobic field retting is preferred, where hemp stems are left on fields after cutting and exposed to the action of microbes for approximately 2 weeks to 10 weeks (Henriksson et al. 1997). However, despite a growing number of studies on the physical and chemical property changes that occur during retting, there is a lack of knowledge on the nature and activities of microbes and the resulting micro-morphological and ultrastructural changes that occur in hemp stems and bast fibres during this process.

Hemp bast fibres are produced through the physical and chemical breakdown of the stem cellular structure achieved by the collective action of fungi and bacteria. However, the two microflorae, while similar to that formed in other naturally decomposing habitats (e.g., litter environments), have major differences in their microbial biomass nutrient demands, which should influence decomposition, including the use of cellular plant materials as a carbon source (Sinsabaugh et al. 2009; Keiblinger et al. 2010). Recent research has revealed the coexistence of bacteria and fungi in many natural environments that leads to bacterial-fungal interactions (BFIs). These interactions can render both benefits and/or antagonisms within the habitat and are shown to form physical and metabolically interdependent consortia that collectively affect the habitat, as shown by litter decomposition (Tarkka et al. 2009; Frey-Klett et al. 2011). Field retting is contemporary with other natural environments, but no attempt has been made to identify BFIs that exist during hemp retting and thereby whether they are responsible for regulating partial decomposition of hemp stems.

The objectives of the present work were to obtain a more comprehensive understanding of field retting mechanisms concerning micro-morphological and ultrastructural aspects, and identify BFIs that could impact the retting and quality of bast fibres. Molecular identification of the microbial community and an in-depth study using scanning electron microscopy (SEM) were performed on freshly harvested and field-retted hemp stems over a period of 10 weeks to gain information on the microbial colonization and development, and progressive morphological and ultrastructural changes that occurred in the hemp stems and bast fibres. To the best of our knowledge, our study represents the first comprehensive study on hemp field retting that revealed coexisting interactions of BFIs related to progressive microbial break down of plant materials permitting extraction of bast fibres. This study will be able to provide important clues for hemp/plant fibre industries that utilize retting processes for obtaining reinforcing fibres that have a constant quality and high yield, and may also assist in averting negative impacts on the environment. Such knowledge can provide an important foundation for future studies on field retting, suggesting better implementation of management practices: speeding up the process while reaching optimum degree of retting and efficient removal of bast fibres with better-preserved fibre properties.

EXPERIMENTAL

Plant Materials

Hemp (Cannabis sativa L.) variety USO-31 was grown in France (planted on 5 May 2013) and harvested in the early (flowering stage; 18 July 2013) and late (seed maturity; 6 September 2013) growth stages based on the definition of Mediavilla et al. (1998) for code 2101 and 2204, respectively. The details of the growing conditions, harvesting, etc. can be found in Liu et al. (2015). Hemp stem samples for the study were randomly selected from the two harvests.

The early harvest stems were subjected to 50-d and 70-d retting periods and the late harvest stems were subjected to 7-d, 14-d, and 20-d retting periods. For more details, consult Liu et al. (2015). After field retting, the samples were kept frozen until the laboratory and microscopy studies were conducted. Table 1 summarizes all sample types used and their respective analyses.

Table 1: Summary of Different Hemp Stems Sample Types Used in the Study and Relevant Properties/analysis Performed Including Treatments Before the Respective Analyses

^a Retting period in days (sampling point)

To remove fungal materials from the stem surfaces (after retting) and remove bast fibres from the rest of the woody core, both control and retted hemp stem samples were washed in water and manually peeled, except for the stem samples used for the microscopy studies. The resulting bast fibres were then dried at 50 °C for 24 h before the chemical analyses described below.

Dry Matter and Compound Recovery

The dry matter (mass loss) and elemental composition of the fibres were calculated to assess the degradation of the hemp bast fibres from field retting for different time periods. The chemical composition of the dried bast fibres (50 °C for 12 h) was analysed after grinding them with a microfine grinder (IKA MF 10.1, IKA^® -Werke GmbH, Staufenim Breisgau, Germany) and Soxhlet extracting (Gerhardt EV6 ALL/16 No. 10-0012, C. Gerhardt GmbH & Co. KG, C., Königswinter, Germany) the fibre particles approximately 1 mm in size using a toluene-ethanol-acetone (4:1:1 v/v) solution to remove the extractives. Following acid hydrolysis of the extractive-free fibres, chemical analyses of monosaccharides were done using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Dionex ICS-3000, ThermoFisher Scientific, Roskilde, Denmark). The Klason lignin was measured gravimetrically, as described by Liu et al. (2017a). The recovery values for the monosaccharides were estimated from the parallel runs, as described by Arnous and Meyer (2008). The composition of the major hemp fibre polymers was determined, including the cellulose (glucose), pectin (galacturonic acid (GalA), galactose, arabinose, and rhamnose), hemicellulose (xylose and mannose), and Klason lignin contents.

The initial recoveries of the dry matter after field retting (early harvest: 50 d of retting; late harvest: 7 d and 14 d of retting) were estimated by assuming zero degradation of the lignin and cellulose, which can be justified because initially mainly the pectin was degraded according to the compositional changes. After 70 d of retting for the early harvest and 21 d of retting for the late harvest, the dry matter loss was calculated based on the cellulose loss because the concentrations of the other polymers appeared constant or slightly increased. The concentrations used are shown in Figs. 1a and 1b. The following equation was used for calculating the dry matter (DM) recovery during the initial stages by assuming no cellulose and lignin degradation for a time (x, d) less than or equal to the retting time (t₁, d) (t₀ = 0 d and t₁ = 50 d for the early harvest and 14 d for the late harvest):

For the later periods of retting, where mainly cellulose degradation occurred for x greater than t₁, the DM recovery was calculated with the following equation:

The composition of the major polymers (i.e., cellulose, lignin, and pectin) in the samples was calculated relative to the amount of DM when field retting was initiated as recovery (Rec):

Fungal and Bacterial Identification by Gene Sequencing

The hemp bast fibres were isolated manually from the hemp plant stems and characterized for the bacterial and fungal community, as described in detail by Liu et al. (2017a). In brief, fibre samples (2 mm²) were extracted to obtain genomic DNA using the PowerBiofilm™ DNA Isolation Kit (MO-BIO, Carlsbad, USA). Polymerase chain reaction amplification was done using the 16S ribosomal ribonucleic acid primers 27F (5′-AGAGTTTGATCATGGCTCA-3′) and 1492R (5′-CGG TTA CCT TGT TAC GACTT-3′) for bacteria, and ITS5 (5′-GGA AGT AAA AGT CGT AAC AAGG-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) for fungi. Cloning was performed using the pJet1.2/Blunt cloning vector (50 ng/µL) and T4 DNA ligase (5 U/µL), and the ligated product transformed into E. coli DH5α. Purified plasmids were sequenced using the 27F primer for bacteria and ITS4 primer for fungi. A GenBank nucleotide database search was conducted using the BLAST algorithm to identify the sequences. The identified sequences are published in the EMBL Nucleotide Sequence Database with accession numbers LT622055 and LT622085 outlined in Liu et al. (2017a) for bacteria and fungi, respectively.

The fraction of each fungal genus was determined relative to the total number of fungal sequences at each retting time point, while the fractions of bacterial sequences were similarly determined relative to the total number of bacterial sequences. The calculations for bacteria were done based on Liu et al. (2017a) and were as follows: Pseudomonas spp. = Pseudomonas argentinensis + Pseudomonas rhizosphaerae + Pseudomonas syringae; Pantoea spp. = Pantoea agglomerans + Pantoea brenneri; Massilia spp. = M. aurea; and Rhizobium spp. = R. soli. The calculations for the fungi were done based on Liu et al. (2017a) and were as follows: Alternaria spp. = A. brassicae + A. infectoria; Cladosporium spp. = Cladosporium antarcticum + Cladosporium macrocarpum + Cladosporium uredinicola; Stemphylium spp. = S. globuliferum; and Cryptococcus spp. = Cryptococcus carnescens + Cryptococcus festucosus spp. + Cryptococcus victoriae spp.

Scanning Electron Microscopy

Never-dried hemp stem samples that were field-retted and control samples (i.e., non-retted) for the SEM investigations were taken either directly after treatment or thawed to room temperature if they were stored frozen after harvesting (under -20 °C). Stem sections (5 cm × 2 cm) were cut from the hemp stem samples using stereomicroscopy for the surface and cross-sectional observations for each treatment and control sample. The sections were fixed in a mixture of 3% v/v glutaraldehyde and 2% v/v paraformaldehyde in 0.1 M sodium cacodylate buffer (pH = 7.2) for 4 h at room temperature. After three washes in 0.1 M buffer and two washes in water (10 min, each), the samples were dehydrated in a series of solutions with an increasing ethanol concentration (20% to 100% with 20% steps for 10 min, each), followed by acetone (3:1, 1:1, and 1:3 ethanol:acetone, and finally 100% acetone for 10 min, each). The samples were subsequently critical point dried in an Agar E3000 critical point dryer (Quorum Technologies Ltd., East Essex, UK) using CO₂ as the drying agent, mounted on stubs using double-sided tape, and coated with gold using an Emitech K550X sputter device (Emitech Ltd., Kent, UK). The observations were made using a Philips XL 30 ESEM (FEI, Eindhoven, Netherlands) operated at 10 kV to 15 kV, and the images were recorded digitally.

RESULTS AND DISCUSSION

Mass Loss and Lignocellulose Degradation

The mass recovery reached similar values (84%) during both the early (i.e., 70 d) and late harvests (i.e., 21 d) (Figs. 1c and 1d). The slow mass loss rate after the early harvest presumably resulted from the high daily temperatures with low precipitation during August, which resulted in faster evaporation (Liu et al. 2015). Hence, the dryer hemp stems had a lower microbial activity and required an extended field retting duration. Initially, pectin degradation occurred during both the early and late harvests (Figs. 1c and 1d) with a higher rate during the late harvest, followed by cellulose degradation that was more pronounced after 50 d of retting (Fig. 1c). Lignin with a predominance of guaiacyl (G) units (syringyl (S)/G ratio of 0.8) (del Río et al. 2007) was recalcitrant to microbial degradation and accumulated over the entire retting period (Figs. 1a, 1b, 1c, and 1d). However, the lignin content of the hemp fibres was low and thus the total lignin only marginally increased. The level and type of lignin in wood cell walls is generally considered to be a major factor that influences wood decay, with G-rich lignified fibres more resistant to decay than those containing S-rich lignin (Faix et al. 1985; Blanchette 1991). This knowledge combined with previous data on similar samples that showed a lack of ligninolytic Basidiomycota fungi, especially during the latter part of hemp field retting (Liu et al. 2017a), explained the observed lignin accumulation.