Effective Use of Flax Biomass in Biorefining Processes

Abstract

Flax is one of the few plants that are entirely a source of raw materials for further production. Promising directions for the use of flax biomass may be the production of bioenergy in the form of 2G biofuels and the production of “green” composites. The aim of the study is to compare the biomass of fiber flax, linseed and dual-purpose varieties of cultivated flax (Linum usitatissimum L.) susceptibility to the biorefining processes. In the first stage of the research, based on the results of yield structure features and biometric measurements of plants, the most optimal flax line was selected for the fiber flax, linseed and dual purpose. Next, the forms of flax were pretreated with sodium hydroxide (NaOH), the chemical composition was determined and SEM images were taken. The obtaining of bioethanol process SFF (simultaneous saccharification and fermentation) was carried out. In addition, biodegradable polymers were modified with flax biomass, shapes were prepared, and the rheological and mechanical properties, as well as microbiological activity of biocomposites, were determined. The highest concentration of ethanol (8.72 g·L⁻¹) and the greatest susceptibility to mold fungi of the biocomposites were obtained for the fiber flax variety PET 16/20.

1. Introduction

Cultivated flax (Linum usitatissimum L.) belongs to the agricultural species with a very wide range of applications. It is an annual plant with medicinal and dietary properties, also used in industry. Three types of flax are distinguished in cultivation forms: fiber flax, linseed and dual purpose [1]. The total cultivated area of flax in the world in 2021 was 4,384,000 ha, of which only 241,103 ha was used for fiber flax [2].

Linseed varieties are focused on a high yield of seeds, which are characterized by the highest content of alpha-linolenic acid among plants [3,4]. Flax seeds contain 33–45% fat, and linseed oil over 50% of highly saturated fatty acids—mainly alpha-linolenic acid (omega 3) and linoleic acid. The main producers of linseed are Russia (1,492,119 ha), Kazakhstan (1,366,068 ha) and Canada (403,500 ha) [2]. Flax seeds are used in the food and baking industry (bread, biscuits and breakfast cereals), the oil industry (edible oil, paints, varnishes, printing inks), the pharmaceutical industry and as dietary supplements.

The basic raw material obtained from the cultivation of fibrous forms is straw and fiber. Flax straw is used to obtain long fiber used to produce high-quality yarns and fabrics, and short fiber, which is used to produce carded yarns, insulating mats or paper [5,6]. Fiber flax is mainly cultivated in France (112,580 ha), Belarus (42,300 ha) and Russia (36,483 ha) [2].

In addition, to increase the profitability of flax cultivation, breeding works are carried out to produce dual-purpose varieties [7,8,9]. It is an attempt to combine a high yield of seeds with a high yield of monomorphic fiber, intended for technical purposes.

However, the fact that natural bast fibers constitute a negligible part of the global textile market makes it difficult to sell basic raw materials obtained from flax, especially fiber. The main reason for this is the replacement of flax fiber with cotton and synthetic fibers, and the high cost of natural fiber products. Moreover, weather conditions have a great influence on the quality of the fiber. All this makes it necessary to develop alternative directions for the use of flax biomass [10]. One of them is the use of flax biomass in biorefining processes.

Lignocellulosic biomass is the oldest and most widespread source of renewable energy, the third largest natural energy source in the world. The use of biomass is inextricably linked to the reduction of greenhouse gas emissions, improvement of energy security and socio-economic development.

The cultivation and processing of flax plants can be climate-neutral and harmoniously integrated into a circular bioeconomy. As mentioned, thanks to the multi-directional use of flax raw materials, by-products do not have to be waste, but a raw material for further production.

In this study, one of the planned biorefining processes of flax biomass is the process of obtaining 2G bioethanol, because according to the assumptions of the RED II Directive, it introduced the obligation to reach 14% share of RES in transport by 2030, including at least 3.5% from advanced biofuels (from non-food plant matter) [11].

The structure of plant biomass is formed by lignocellulose, in which cellulose and hemicellulose are potential substrates in the process of obtaining biofuels, but lignin consisting of aromatic compounds is a strong obstacle in this process.

Therefore, there is a need to carry out pretreatment, the purpose of which is to loosen the compact structure of lignocellulose and delignify the raw material [12]. The next stages of processing plant biomass into bioethanol are enzymatic hydrolysis and ethanol fermentation. An effective and economical solution seems to be the SSF process combining cellulose hydrolysis with sugar fermentation in one bioreactor [13,14,15].

Natural plant raw materials often contain substances that have antibacterial or antifungal properties, which reduces the number of microorganisms and helps to keep them at a safe, low level.

The current market, and especially the requirements of today’s customers, prefers a hygienic lifestyle and attaches more and more importance to the issue of health protection and care for the natural environment. In addition, due to legal regulations, environmentally friendly materials are increasingly sought by manufacturers of industrial products and their users [16,17].

The plastics industry is looking for new materials that will reduce the amount of waste. Both legislation and consumer expectations require less environmental impact from plastics. This increases interest in environmentally friendly biodegradable materials. One of the leading biodegradable polymers on the market is polylactic acid (PLA) in the packaging industry [18,19,20].

However, in many areas, the increased use of biodegradable materials is limited by their higher price than currently used polymers. A way to reduce the consumption of valuable biodegradable polymers may be to replace some of them with cheaper biomass of annual plants, e.g., from flax or hemp [21,22].

Considering that, the use of natural fillers for polymer matrices requires suitable preparation to obtain biocomposites with good mechanical properties [23]. Increased production of biocomposite materials in the EU will result in growing interest from industry for components based on renewable raw materials, including flax biomass and fibers. All this contributes to the great interest in research on the mechanism and properties of PLA composites with natural fillings derived from various plant materials [24,25,26,27].

Overview studies on the production of bioethanol from lignocellulosic biomass and the authors’ experience in this scope [28,29], allow us to say that the conducted research is an important element in the search for an alternative method of flax straw management to expand the sale of the raw material and increase the profitability of crops. So far, there are no literature reports describing the use of flax biomass in the biorefining process.

Therefore, the aim of this presented study is the compared perspective lines of cultivated flax—linseed (LS), fiber flax (FF) and dual purpose (DP), obtained by crossing selected parental forms, in terms of the efficiency of the bioethanol production process and the functional and microbiological properties of biocomposites with flax biomass.

2. Materials and Methods

2.1. Flax Biomass

The cultivated flax biomass was obtained from the Experimental Farm of the Institute of Natural Fibres and Medicinal Plants National Research Institute in Pętkowo, Poland.

The research material consisted of 12 homogeneous lines of three functional forms of flax (

Table 1. List of flax lines tested in the experiment.

No.	Flax Lines	Cultivation Form
1	PET 16/02	Linseed
2	PET 16/06	Linseed
3	PET 16/07	Linseed
4	PET 16/16	Linseed
5	PET 16/21	Linseed
6	SW 16/01	Dual purpose
7	SW 16/05	Dual purpose
8	SW 16/08	Dual purpose
9	SW 16/13	Dual purpose
10	PET 16/20	Fiber flax
11	PET 16/09	Fiber flax
12	PET 16/23	Fiber flax

).

The breeding lines were obtained by controlled pollination of the parental forms then, the best single plants for reproduction were selected from the obtained genotypes. Genetically stabilized lines of the F₇ generation were used for the study.

The flax biomass obtained for the tests was prepared according to Figure 1.

2.2. Cultivation of Flax

Plants of the tested flax lines were grown in a pot experiment in a greenhouse. Each line was plated in five replications (5 pots). The pots were filled with soil from the experimental field of valuation class IV b, then they were placed randomly. Standard mineral fertilization was used for the cultivation of flax. A total of 30 seeds were sown in each pot. Seed sowing was carried out in the second half of April 2021. Flax plants were harvested at the stage of full seed maturity in the first half of August.

2.3. Biometric Measurements of Flax

The assessment of the traits that have the greatest impact on the biorefining processes efficiency—straw yield, total plant yield, plant height and technical plant length, was the basis for the selection process. Seed weight and panicle length were also determined. The total yield was determined by weighing the harvested plants after removing the roots and then plants were ginned to determine straw yield. To determine the total length of the plant, it was measured from the beginning of the roots to the top of the panicle, whereas measuring the plant from the root collar to the first branching of the panicle will allow you to determine the technical length. A total of 20 randomly selected plants from each repetition were used for biometric measurements (see Section 2.2). The results are the average of a combination of 5 replicates.

In addition, in order to fully evaluate plants, for the three best lines, one from each variety of flax, an analysis of the total fat content was carried out. Chemical analysis of fat content was performed in a Soxhlet extractor. After grinding, samples of flax were degreased for 3 days, ground and additionally degreased for 1 day. Each sample was analyzed in duplicate.

2.4. Bioethanol Production Process

2.4.1. Flax Biomass Pretreatment

The flax raw material was crushed into particles of 20–40 mm and dried for 24 h at 50–55 °C. In the next stage, the biomass was ground using a cutting mill (Retsch SM-200, Haan, Germany) into 2 mm fragments. The crushed raw material was chemical pretreatment with 2% sodium hydroxide for 5 h at 90 °C [28]. The weight ratio of NaOH to biomass was 10:1. To neutralize, the alkaline solution of the tested biomass was filtered and washed and dried for 24 h at 50 °C. Then, using the Miller method, the amount of released reducing sugars obtained after the alkaline pretreatment was determined [30]. The enzyme test was performed using Flashzyme 200 (AB Enzyme, Darmstadt, Germany) at a dose of 20 FPU·g⁻¹. The flax biomass was incubated at 55 °C in 0.05 M citrate buffer of pH 4.8 for 24 h. After this test, the supernatant was appropriately diluted, DNS acid was added and the mixture was incubated in a boiling water bath for 10 min. Then, the supernatant was cooled to room temperature and the absorbance at 530 nm was measured (UV-VIS spectrophotometer, Jasco V-630, Pfungstadt, Germany).

2.4.2. Process of Simultaneous Saccharification and Fermentation (SSF)

The SSF process was carried out in 100 mL flasks and the total volume of flax biomass hydrolysate was 40 mL. The prepared solution was subjected to pH adjustment to the desired value (pH 4.8) using 1 M sulfuric acid and 1 M sodium hydroxide. Then Flashzyme Plus 200 (AB Enzyme) enzyme was added in the amount of 20 FPU·g⁻¹ and non-hydrated freeze-dried yeast Saccharomyces cerevisiae was added in the dose of 0.5 g·L⁻¹ (corresponding to the cell concentration after inoculation of about 1 × 10⁷ cfu·mL⁻¹). The flasks (plugged with stoppers with fermentation tubes) were incubated at 37 °C for 72 h on a shaker (200 rpm).

2.5. Biocomposites Production Process

2.5.1. Natural Fillers from Halophyte Biomass

Samples of three forms of flax biomass were dried and ground in a Rekord A mill (Jehmlich, Nossen, Germany) with a sieve separator with a mesh size of 0.5 mm. A sieve analysis of the obtained natural fillers was performed using the Analysette 3 Spartan analyzer (Fritsch, Idar-Oberstein, Germany) and their moisture content was determined with a MA.X2.A moisture analyzer (Radwag, Radom, Poland).

2.5.2. Polymer Matrix

A biodegradable polymer—poly(lactic acid) of the Ingeo 3251D variety (Nature Works, Plymouth, MN, USA) with a density of 1.24 g·mL⁻¹ and a melt flow rate (MFR) of 35 g/10 min (190 °C, 2.16 kg) was used as the matrix for the biocomposites.

2.5.3. Composites Preparation

PLA blends with 20% biomass content of flax varieties designated as PLA-FF-20, PLA-DP-20 and PLA-LS-20 were compounded in co-rotating twin screw extruder Leistritz MICRO 27 GL/GG-44D (Leistritz Extrusionstechnik, Nürnberg, Germany) with Brabender gravimetric feeding system (Brabender Technologie, Duisburg, Germany). Compounding conditions: barrel temperature profile 170–200 °C, extrusion speed of 150 rpm and throughput 16 kg·h⁻¹. Then, the composite granules were dried to a humidity level below 0.05% in a Drywell DW25/40 molecular dryer (Digicolor, Herford, Germany) at a temperature of 70 °C (dew point −40 °C).

In order to determine the properties of the obtained biocomposites and microbiological tests, type 5A test samples in accordance with ISO 527-2 were molded by injection molding machine Haitian Mars MA600 (Haitian Plastics Machinery, Ningbo, China) [31]. Temperature profiles were 180 °C (hopper), 185 °C, 190 °C and 190 °C (nozzle). The temperature of the mold was set to 40 °C.

2.5.4. Resistance of Biocomposites to Mold Fungi

The samples of polylactide biocomposites with 20% biomass content from three varieties of flax: PLA-LS-20, PLA-DP-20 and PLA-FF-20 and samples of pure polylactide—PLA 3251D as references were used for the test of resistance to mold fungi. The tests were carried out in accordance with the methodology specified in the standard ISO 846 [32]. The tested samples were allocated on an agar medium and inoculated with a mixture of five strains of mold fungi, which most often cause the decomposition of cellulose: Aspergillus niger, Chaetomium globosum, Penicillium ochrochloron, Paecilomyces variotii and Penicillium funiculosum. The agar medium with mineral salts was used, prepared in accordance with the standard, using Chempur reagents and agar powder from Merck, whereas tested microorganisms originated from the pure culture collection of the Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Poland.

Then, the inoculated samples were placed in a climatic chamber in optimal conditions for mold development, i.e., at a temperature of 29 ± 1 °C and relative air humidity of 90% for 28 days. These samples were designated as series I. For comparison, samples of biocomposites in each variant, which were allocated on a sterile agar medium and not inoculated with a mixture of mold fungi, were placed in the same climatic conditions. These samples were designated as series S. In the case of samples not inoculated with the test fungi, the degree of infection with microorganisms other than the test fungi was assessed. In addition, biocomposite control samples for each variant, not incubated, were left and stored under standardized conditions of room temperature and relative humidity. These samples were designated as series 0.

After 28 days, visual assessment of fungal growth on the samples’ surfaces was assessed on a scale from 0 to 5, where:

• 0—no visible growth under the microscope
• 1—growth invisible to the naked eye but clearly visible under the microscope
• 2—growth visible to the naked eye, covering up to 25% of tested surface
• 3—growth visible to the naked eye, covering up to 50% of tested surface
• 4—considerable growth, covering more than 50% of tested surface
• 5—intensive growth, covering all tested surface.

Next, the samples were cleansed and mechanical properties were evaluated. All the measurements were performed six times.

2.6. Analytical and Testing Methods

The chemical composition of the main cultivated flax biomass components before and after alkaline pretreatment was determined according to standard methods. The cellulose content was determined by the Seifert method using a mixture of acetylacetone and dioxane [33]. The lignin content was determined by the Tappi (Technical Association of the Pulp and Paper Industry) method (T-222 om-06) using concentrated sulfuric acid [34]. The theoretical hemicellulose content was arithmetically calculated as the difference between holocellulose (TAPPI—T9 wd-75) and cellulose [35]. All chemical composition results were the average of three measurements and were calculated on the basis of the dry weight of the raw material.

In addition, for flax biomass before and after pretreatment, using a scanning electron microscope (SEM, S-3400N, Hitachi, Tokyo, Japan) under high vacuum conditions, the physical morphology was determined. Biomass samples were covered with gold dust.

The ethanol concentration from flax biomass was determined by HPLC on Elite LaChrom liquid chromatograph from VWR-Hitachi using an RI L-2490 detector, Rezex ROA 300 × 7.80 mm column from Phenomenex, at a flow rate of 0.6 mL·min⁻¹, at 40 °C. The samples were loaded onto the column at 10 µL. The quantitative identification was performed by the external standard method using the peak area (measurement and computer integration using the Ez-Chrom Elite program).

Mechanical properties tests of the composites were carried out at room temperature with a universal testing machine Inspekt Table 50 (Hegewald & Peschke MPT, Nossen, Germany) according to ISO 527 and ISO 178, respectively [31,36,37]. In both tests, the crosshead speed was set to 5 mm·min⁻¹. Tensile tests in accordance with ISO 527-1 and 2 were carried out using an MFA clip-on extensometer (MF Mess- & Feinwerktechnik, Velbert, Germany) with a nominal length of 20 mm [31,36].

2.7. Statistical Analysis

The SSF process experiments were performed in triplicate. Standard ethanol concentration deviations were calculated using ANOVA analysis of variance, Statistica 13.0 software (p < 0.05) [38].

3. Results and Discussion

3.1. Selection of Optimal Genotype for the Fiber Flax, Linseed and Dual Purpose

A comparative analysis of selected lines of flax with a high degree of homozygosity was carried out in order to select the best genotypes for biorefining processes.

The lines were compared in terms of the following yield-forming features: total yield, straw yield, seed yield, plant height, technical length and panicle length. The test results are presented in

Table 2. Comparison of tested linseed lines in terms of yield structure traits.

Breeding Line	Total Yield	Straw Yield	Seed Yield	Plant Height	Technical Length	Panicle Length
	(g)	(g)	(g)	(cm)	(cm)	(cm)
PET 16/02	146.4 ± 47.3	76.4 ± 25.6	48.0 ± 11.4	50.5 ± 3.1	39.3 ± 2.0	11.2 ± 2.5
PET 16/06	126.4 ± 22.5	52.8 ± 5.2	31.6 ± 9.8	46.3 ± 2.5	36.6 ± 1.9	9.7 ± 2.9
PET 16/07	92.8 ± 21.9	32.4 ± 17.7	31.6 ± 9.8	42.5 ± 2.8	33.9 ± 1.5	8.6 ± 1.5
PET 16/16	200.4 ± 16.6	79.6 ± 17.8	70.0 ± 4.5	50.0 ± 2.7	40.5 ± 2.8	9.4 ± 1.4
PET 16/21	111.2 ± 28.2	48.8 ± 11.5	28.8 ± 9.9	47.6 ± 5.1	36.3 ± 4.6	11.2 ± 1.7

,

Table 3. Comparison of tested dual-purpose lines in terms of yield structure traits.

Breeding Line	Total Yield	Straw Yield	Seed Yield	Plant Height	Technical Length	Panicle Length
	(g)	(g)	(g)	(cm)	(cm)	(cm)
SW 16/01	153.6 ± 25.3	57.2 ± 16.2	52.8 ± 14.8	43.7 ± 8.6	33.4 ± 7.5	10.0 ± 1.9
SW 16/05	123.2 ± 41.7	48.8 ± 18.0	48.0 ± 16.0	39.4 ± 3.6	29.7 ± 3.2	9.6 ± 1.4
SW 16/08	120.8 ± 23.8	50.8 ± 14.3	38.8 ± 5.4	46.0 ± 2.0	38.0 ± 2.4	8.0 ± 1.7
SW 16/13	143.6 ± 19.9	64.0 ± 14.6	48.8 ± 8.3	44.6 ± 6.6	35.0 ± 4.0	9.5 ± 2.6

and

Table 4. Comparison of tested fiber flax lines in terms of yield structure traits.

Breeding Line	Total Yield	Straw Yield	Seed Yield	Plant Height	Technical Length	Panicle Length
	(g)	(g)	(g)	(cm)	(cm)	(cm)
PET 16/20	122.8 ± 14.3	70.4 ± 8.5	36.8 ± 3.3	50.9 ± 3.0	41.7 ± 1.7	9.2 ± 1.6
PET 16/09	113.2 ± 28.2	70.0 ± 22.9	28.4 ± 8.7	67.6 ± 5.2	50.4 ± 5.4	17.2 ± 1.3
PET 16/23	125.2 ± 10.1	66.8 ± 11.5	37.6 ± 6.2	53.4 ± 3.2	41.2 ± 4.1	12.2 ± 2.6

.

A significant differentiation of the tested linseed lines was found in terms of yield-forming features, i.e., total yield, straw yield and seed yield. The PET 16/16 line was characterized by significantly the highest total plant weight and seed weight compared to the other tested linseed genotypes. The straw weight of this line was also the highest, with significant differences observed in comparison to the lines: PET 16/06, PET 16/07 and PET 16/21.

There was definitely less differentiation between the examined objects observed in the case of morphological traits, i.e., plant height, technical length and panicle length.

For biomass biorefining processes, however, straw yield and total yield are the most important, and on this basis, the best line was PET 16/16.

The differentiation of the tested dual-purpose lines in terms of yield-producing traits was lower than in the case of linseed lines. The highest total plant weight was found in line SW 16/01, whereas the highest mass of straw was characterized by line SW 16/13. The straw weight of the SW 16/13 line was 10.6% higher than the straw weight of the SW 16/01 line, with the total weight of the plants being lower by 6.6%. However, since the mass of straw is considered to be the most important trait for biomass biorefining processes, a line SW 16/13 was selected for further chemical research.

Within fiber flax lines, very slight differences in the tested objects in terms of straw weight were observed. Therefore, the most important trait did not differentiate the compared lines to a sufficient extent to make a clear choice of the best one on this basis.

The highest straw weight was found for line PET 16/20, but line PET 16/09 showed only 0.4 g less weight of straw. However, the line that showed the highest straw yield (PET 16/20) was also characterized by a slightly lower total yield than the best line (PET 16/23), and significantly higher than the line PET 16/09. On this basis, it was decided that the line PET 16/20 would be directed for further chemical research.

Summing up, in this study, the straw yield was significantly highest among the fiber flax lines (an average of 69 g), and the lowest within the dual-purpose lines (an average of 55.2 g). However, dual-purpose lines were characterized by a slightly lower mass of straw than linseed lines (an average of 58 g). In turn, the seed yield was the highest in dual-purpose forms, slightly lower in linseed forms, and significantly lowest in fiber flax forms.

Since it is possible to effectively use both straw and seeds in the cultivation of flax, the total fat content in the seeds of three selected lines of flax was also analyzed (

Table 5. Comparison of selected lines of flax in terms of fat content in seeds.

No.	Breeding Line	Fat Content (%)
1	PET 16/16	30.9 ± 0.4
2	SW 16/13	31.1 ± 0.6
3	PET 16/20	28.5 ± 0.3

).

The highest fat content was found in the dual-purpose line SW 16/13. In this respect, it exceeded not only the fiber flax form (PET 16/20) but also the linseed form (PET 16/16). Whereas, fat content in the seeds of linseed forms is higher compared to fiber flax forms, which is confirmed by numerous studies [39,40]. However, the difference between the best line SW 16/13 and the worst line PET 16/20 was only 2.6%. This proves the high lability of the fat content in flax seeds and the possibility of profitable use of seeds, regardless of the cultivation form.

3.2. Bioethanol Production Process

3.2.1. Flax Biomass Pretreatment

Physicochemical pretreatment was carried out, which is a very important stage in the conversion of biomass for energy purposes. The effect of the process is to loosen the compact structure of lignocellulose, which enables better access of enzymes to cellulose, increasing the susceptibility of biomass to decomposition into simple sugars, which are then converted into ethanol in the fermentation process.

The selected flax line’s biomass was ground using a knife mill on a sieve with a mesh of 2 mm. The biomass was treated with 2% sodium hydroxide and the content of released reducing sugars was determined using the Miller method (Figure 2).

It was found that the highest content of reducing sugars was obtained for the form of fiber flax—the line PET 16/20, which amounted to 248.1 mg·g⁻¹. The linseed turned out to be the most alkali resistant. However, the difference between these forms was not too large and amounted to 27.3 mg·g⁻¹, which was 11% of the value obtained for fiber flax.

Next, the chemical composition of three varieties of flax biomass was assessed, before and after alkaline treatment using chemical methods (

Table 6. Chemical composition of flax biomass before (BP) and after (AP) alkaline treatment (%).

Cultivation Form	Sample	Cellulose	Holocellulose	Hemicellulose	Lignin
Linseed	BP	37.42 ± 0.62	69.31 ± 0.99	31.89 ± 0.41	17.83 ± 0.43
	AP	47.82 ± 0.17	71.61 ± 0.39	23.79 ± 0.51	20.30 ± 0.60
Dual purpose	BP	39.94 ± 0.21	72.13 ± 0.26	32.19 ± 0.42	18.44 ± 0.10
	AP	49.20 ± 0.56	72.31 ± 0.44	23.11 ± 0.54	21.26 ± 0.04
Fiber flax	BP	42.17 ± 0.29	74.27 ± 0.47	32.10 ± 0.63	16.93 ± 0.31
	AP	51.40 ± 0.58	75.72 ± 0.21	24.32 ± 0.55	19.86 ± 0.30

).

The fiber flax has the highest cellulose content, which is confirmed by the results of the content of reducing sugars. Alkaline treatment resulted in partial degradation of hemicellulose. The seeming increase in lignin content was mainly due to the loss of hemicellulose as a result of the alkaline pretreatment environment.

Similar values of chemical composition were observed for the biomass of other plants such as hemp. Before chemical treatment, cellulose was in the range of 47–51%, hemicellulose was equal to 28–33% and lignin was 14–16%. Whereas, hemp biomass subjected to chemical treatment with NaOH was characterized by an increased content of cellulose (57–63%), as well as partial degradation of hemicellulose (20–22%). In turn, the lignin content, similarly to flax biomass, slightly increased after chemical treatment (17–18%) [29]. On the other hand, for sorghum biomass treated with 2% sodium hydroxide, an increase in the cellulose content from 37% (before pretreatment) to 71% was observed, and a decrease in the content of hemicellulose from 25% to 16%, and especially lignin from 21% to 6% [41].

SEM photos of biomass were also taken for all forms of flax before and after treatment with 2% NaOH (

Table 7. SEM photos of flax biomass before and after pretreatment.

	Before Pretreatment	After Treatment with 2% NaOH
Dual purpose
Linseed
Fiber flax

).

Untreated flax biomass is intact and has a sedimentary layer on the surface that effectively blocks access to lignocellulose [42]. After treatment with NaOH, all tested varieties of flax, especially fiber flax and dual purpose, show damage to the biomass structure and partial cleaning of its surface [43]. This proves the effectiveness of the alkaline treatment and has a positive effect on the enzymatic availability and digestibility of biomass in the subsequent stages of the bioethanol production process.

3.2.2. SSF Process of Flax Biomass

The biomass of selected flax lines was subjected to the SSF process for 72 h at 37 °C, i.e., in conditions appropriate for the synergy of the Flashzyme Plus 200 enzyme and Saccharomyces cerevisiae distillery yeast. Figure 3 shows the ethanol concentration determined by HPLC.

The highest ethanol content was obtained for fiber flax biomass and it was 8.72 g·L⁻¹, and the lowest for linseed was 7.65 g·L⁻¹ (a difference of about 12% compared to the value of fiber flax). Moreover, the difference between linseed and dual purpose (8.18 g·L⁻¹) was about 0.5 g·L⁻¹, the same as between dual purpose and fiber flax.

A similar ethanol concentration was obtained for hemp biomass, which was pretreated with sodium hydroxide of the same concentration, i.e., 2%, and then the SSF process with the addition of the Flashzyme Plus 200 enzyme. The ethanol content for hemp biomass was in the range of 6–7 g·L⁻¹ [29]. In other studies the SSF was carried out on the pretreated Miscanthus species and the ethanol concentration from this biomass was 7.42 g·L⁻¹ [44].

Summing up, the obtained ethanol concentrations for flax biomass are at a satisfactory level, and the biomass of each selected line of flax can be a potential raw material for obtaining bioethanol.

3.3. Biocomposite Production Process

3.3.1. Fillers from Flax Biomass

Natural fillers from flax biomass with particles smaller than 0.5 mm were prepared. The humidity was determined and a sieve analysis of the natural fillers was performed. The details are shown in

Table 8. Particle size distribution and humidity of fillers from flax biomass.

Cultivation Form	Humidity (%)	Particle Size Distribution (%)
		Below 0.5 mm	Below 0.25 mm	Below 0.2 mm	Below 0.1 mm
Fiber flax	6.8	37	35	23	5
Dual purpose	6.5	31	27	35	7
Linseed	7.2	28	33	30	9

.

The bulk density of fillers from selected forms of flax was also determined and the following values were obtained, i.e., for fiber flax 0.165 kg·L⁻¹, dual purpose 0.172 kg·L⁻¹ and linseed 0.183 kg·L⁻¹.

3.3.2. Mechanical Properties of Biocomposites

The effects of the selected flax varieties biomass on both the tensile and flexural properties of PLA composites are shown in

Table 9. Tensile and flexural properties of PLA/flax composites.

Sample	Tensile Strength δM (MPa)	Tensile Modulus Et (GPa)	Flexural Strength δfM (MPa)	Flexural Modulus Ef (GPa)
PLA 3251D	65.5 ± 1.3	3.3 ± 0.2	105.4 ± 1.0	3.4 ± 0.1
PLA-FF-20	55.6 ± 1.0	5.3 ± 0.3	89.2 ± 2.4	5.6 ± 0.2
PLA-DP-20	54.2 ± 1.2	5.1 ± 0.3	85.7 ± 1.8	4.9 ±0.3
PLA-LS-20	53.1 ± 1.1	5.1 ± 0.3	84.4 ± 1.8	4.6 ± 0.2

.

The use of the PLA composites of flax fillers in the amount of 20% resulted in a decrease in tensile strength in the range of 15–19%. Compared to PLA, the flexural strength of the composites decreased in the range of 13–19%. On the other hand, depending on the variety of linen, an increase in modulus was noted during the elongation (54–60%) and bending (35–64%) of the compositions in relation to PLA.

Natural raw materials containing cellulose, hemicellulose, lignins and pectins are active hydrophilic fillers [45]. The decrease in tensile and flexural strength indicates that despite the use of hydrophilic flax biomass in combination with a hydrophilic biodegradable polymer (PLA), the structure of the biocomposite does not show full adhesion at the interface of individual components [46]. The reason for this may be the different content of fiber and shives in the biomass unit or the different content of cellulose in the composition for different varieties of flax, which is responsible for the effectiveness of adhesion with the biodegradable polymer matrix. The filler/matrix adhesion is an important factor affecting the final mechanical properties of composites. Good interface adhesion ensures effective stress transfer between the matrix and natural filler [27,47].

3.3.3. Microbiological Properties of Biocomposites

In the mold fungal test, regardless of the type of linen biomass, the growth of test fungi was observed on the surface of the tested samples inoculated with microorganisms (series I). The results of the test are shown in Figure 4.

The highest mold fungi resistance was found for biocomposites PLA-LS-20—fungal growth was insignificant (second degree), in the form of single traces of fungi on the surface of the samples. Biocomposites PLA-FF-20 were the most susceptible to the test fungi. Intensive fungal growth (fourth degree) was observed on the samples, covering more than 50% of the tested surface. On the samples of PLA composites without the addition of biomass (PLA 3251D), inoculated with molds, a slight increase was found (second degree), covering up to 25% of the tested surface.

Composites not inoculated with molds (series S), both PLA-LS-20, PLA-DP-20 and PLA-FF-20 showed susceptibility to microorganisms other than test fungi in the second degree, which indicates the presence of substances that are a medium for the growth of microorganisms. The samples of biocomposites PLA-FF-20 were the most susceptible, and the PLA-LS-20 were the least susceptible; however, for all samples it was growth visible to the naked eye, covering up to 25% of the tested surface. At the same time, on samples of PLA composites without the addition of biomass (PLA 3251D), not inoculated, trace development of microorganisms other than test fungi was observed, visible only under a microscope.

In addition, changes in the appearance of the tested samples were assessed. Discoloration of the tested samples of biocomposites containing flax biomass was found, whereas distortions and losses on the surface of the samples were not observed.

The conducted microbiological tests showed a greater susceptibility of biocomposites containing flax biomass to the action of mold fungi, in relation to pure PLA. Moreover, in other studies, it was shown that the addition of lignocellulosic fillers, such as conifer bark or coniferous sawdust to PLA composites increased the susceptibility to the mold fungi fouling process [48]. The observed differences are related to the varieties of flax from which the biomass was obtained. Biocomposites PLA-FF-20 were the most susceptible to the test fungi, whereas biocomposites PLA-LS-20 were the most resistant to mold fungi. This may be due to the fact that lignification is much stronger in the stalks of linseed varieties than in the stalks of flax cultivated mainly for fiber.

3.3.4. Mechanical Properties of Biocomposites after Test Mold Resistance

In order to determine the effect of mold fungi on the mechanical properties of the tested biocomposites, tensile and flexural strength tests were carried out again after the completion of the fungal tests. The results of individual studies are presented in

Table 10. Tensile and flexural properties of PLA/flax composites after microbiological testing: O—reference samples; I—samples inoculated with a mixture of mold fungi; S—not inoculated samples subjected to the same climatic conditions as the inoculated samples.

Sample	Tensile Strength δM (MPa)	Tensile Modulus Et (GPa)	Flexural Strength δfM (MPa)	Flexural Modulus Ef (GPa)
PLA 3251D (O)	65.5 ± 1.1	3.3 ± 0.2	105.4 ± 1.0	3.4 ± 0.1
PLA 3251D (I)	64.8 ± 1.3	3.3 ± 1.0	104.2 ± 1.4	3.4 ± 0.5
PLA 3251D (S)	65.4 ± 1.2	3.3 ± 0.6	105.2 ± 1.6	3.4 ± 0.7
PLA-FF-20 (O)	55.6 ± 1.0	5.3 ± 0.3	89.2 ± 2.4	5.6 ± 0.2
PLA-FF-20 (I)	52.4 ± 1.9	5.0 ± 0.7	84.1 ± 2.3	5.3 ± 0.8
PLA-FF-20 (S)	55.2 ± 1.8	5.3 ± 0.9	88.8 ± 2.0	5.6 ± 0.6
PLA-DP-20 (O)	54.2 ± 1.2	5.1 ± 0.3	85.7 ± 1.8	4.9 ± 0.3
PLA-DP-20 (I)	51.5 ± 2.1	4.8 ± 0.7	81.5 ± 1.7	4.7 ± 0.8
PLA-DP-20 (S)	53.8 ± 2.1	5.0 ± 1.0	85.1 ± 1.6	4.8 ± 0.8
PLA-LS-20 (O)	53.1 ± 1.1	5.1 ± 0.3	84.4 ± 1.8	4.6 ± 0.2
PLA-LS-20 (I)	51.2 ± 1.1	4.9 ± 0.6	81.4 ± 2.1	4.4 ± 1.0
PLA-LS-20 (S)	52.8 ± 2.0	5.1 ± 0.9	84.0 ± 1.7	4.6 ± 0.9

.

The strength of flax biomass biocomposites treated with mold over the test period (28 days) decreased in the range of 3.5–7% compared to reference samples. The effect of only climatic conditions on the biocomposite samples did not significantly affect the change in mechanical properties—the changes did not exceed 1%.

Analogous changes in the strength of individual biocomposites can be observed in terms of elastic modules. Samples treated with mold mixtures showed changes in the elastic module in the range of 4–6% compared to reference samples. Changes in the elastic module of non-inoculated samples did not exceed 1.2%.

Regarding the changes in mechanical properties of biocomposites with biomass of different forms of flax, it can be noted that the largest changes are shown in samples with biomass of fiber flax. These changes relate to both the tensile strength and elastic module of the biocomposites. The smallest changes in strength are observed for biocomposite samples with linseed biomass.

4. Conclusions

In this study, five lines of linseed, four lines of dual purpose and three lines of fiber flax were analyzed, and based on the biometric measurements, one genotype for each form of flax was selected, i.e., linseed—PET 16/16, dual purpose—SW 16/13 and fiber flax—PET 16/20. The obtained values of straw weights for all three forms of flax testify to the significant potential of selected genotypes to produce a large amount of raw material for biorefining processes.

In the next stage, the variety of flax with the greatest energy potential was selected. It was found, based on the content of reducing sugars obtained after alkaline pretreatment, that all selected forms of flax can be an effective raw material for bioethanol production, especially fiber flax, for which 8.72 g·L⁻¹ of ethanol was obtained. These results indicate the possibility of expanding the raw material base to produce 2G bioethanol.

Whereas, the biomass fillers of the three tested varieties of flax caused a decrease in the biocomposite’s tensile strength by several percent compared to the biodegradable PLA polymer and a significant increase in the modulus of elasticity in the range of 53–60%. In addition, the susceptibility of biocomposites containing flax biomass to the action of mold fungi in relation to pure PLA was demonstrated. Biocomposites with fiber flax biomass were the most susceptible to the tested fungi and showed the greatest changes in mechanical properties. The lowest susceptibility to test fungi and the smallest changes in strength were observed for samples of biocomposites with linseed biomass. The obtained results can be the basis for the design of modern biocomposites with the assumed properties depending on the target application, e.g., packaging for food products (vegetables, fruit), elements of equipment for public transport vehicles and elements of furniture and interior design used in public places.

On the basis of the conducted research, the possibility of using flax biomass, in particular fiber flax biomass, for valuable bioproducts—bioethanol and “green” composites, and for each tested form of cultivated flax, the possibility of profitable use of the collected seeds was demonstrated.