Utilization of Festuca arundinacea Schreb. Biomass with Different Salt Contents for Bioethanol and Biocomposite Production

Abstract

Soil salinity is one of the particularly important environmental factors, which limits the growth and development of plants, reduces the amount of crops and causes serious economic damage. The study found that Festuca arundinacea can be successfully used for the management and bioremediation of saline habitats, and it is characterized by a very well-developed root system and an average high biomass production of approx. 14.8 Mg∙ha⁻¹, which can be used for industrial applications. Tall fescue biomass from soils with different levels of salinity was used to obtain bioethanol using 1.5% NaOH pretreatment and for the process of simultaneous enzymatic hydrolysis and ethanol fermentation (SSF). It was found that the content of Na⁺ ions in the tall fescue biomass had no significant effect on the amount of ethanol obtained (the average 19.32 g∙100 g⁻¹ of raw material). F. arundinacea biomass was also used as a natural filler to obtain green composites using a biodegradable polylactide (PLA) matrix. It has been shown that tall fescue biomass with the lowest sodium content in biomass (approx. 2.5 mg∙g⁻¹) from an area with high saline (above 6 g NaCl∙L⁻¹) has the smallest effect on reducing the tensile and flexural strength of composites. Moreover, the highest value of bioethanol concentration (21.2 g∙100 g⁻¹ of raw material) for this biomass sample was obtained.

1. Introduction

Soil salinity is a major environmental factor that limits growth and development of plants and reduces the height and quality of crops, especially in arid and semi-arid regions [1]. The area of naturally saline soils in the world is estimated to be about 1 billion ha, while secondary salinization occurs in 77 million ha. As a result of physiological drought (too high osmotic potential of the soil solution), salt stress causes a water deficiency in plant tissues. It inhibits seed germination and seedling growth, as well as reduces the length of roots and shoots [2].

Lignocellulosic bioenergy crops in marginal habitats, including salt-affected soils, can be an interesting alternative to traditionally cultivated plants. These include tall fescue (Festuca arundinacea Schreb., Poaceae), a cool-season C3 perennial, pasture and turf grass growing up to 70–120 cm in height, with large, dense and deeply rooted clumps. It occurs naturally throughout Europe (north to 62° in Scandinavia), in North Africa and Western and Central Asia [3]. F. arundinacea grows on moist meadows, pastures, levees, riverbanks, salt marshes, seashores and cliffs. It is recorded on several types of substrates, usually on poorly drained, alluvial soils [3]. This is a stress-tolerant species with a tendency of apophytism, encroaching on anthropogenic habitats such as roadsides, railway embankments, wastelands, etc. F. arundinacea is a quickly growing plant distinguished by its large biomass production and broad ecological scale, including high tolerance to abiotic stress, waterlogging, salinity, drought and low temperature [4,5,6,7]. In the case of oxygen deficit in the root zone, tall fescue shows the ability to form aerenchyma [8]. This species has also developed both metabolic and physiological mechanisms to counteract drought such as leaf rolling and a rapid stomatal closure [9]. Thanks to a strong root system, it can take water from the soil from a depth of more than 1 m [3].

Literature data have indicated the usefulness of this species in phytoremediation of soils contaminated with heavy metals: lead, cadmium, copper, zinc and nickel [10,11]. These biological features make tall fescue a model plant in research concerning molecular mechanisms of grass tolerance to abiotic stress [12].

Due to low rainfall, high evaporation, salt-water irrigation, overfertilization and other factors, saline areas are increasing by about 10% per year, and we conducted preliminary research on the use of halophytes in the development of an integrated system for bioremediation of saline soils and biorefinery of their biomass [1,13].

Halophyte biomass contains a lignocellulose complex, which is relatively resistant to biodegradation. Biofuel production from plant biomass requires degrading cell walls to specific polymers and hydrolysis of carbohydrates to monomer sugars. It includes the plant material preparation (physico-chemical pretreatment), enzymatic hydrolysis up to alcoholic fermentation [14]. Pretreatment of plant biomass generally allows for loosening of the compact structure of lignocellulose, including increasing the internal surface of the cellulose, reducing the degree of polymerization and crystallinity, and delignification of the raw material [15,16]. Next, an effective and economical solution for further processing of biomass into ethanol seems to be the SSF process, which combines cellulose hydrolysis with fermentation of sugars in one bioreactor, and the enzymes must be adapted to the fermentation temperature of the process of 30–40 °C (optimal temperature for distillery yeast Saccharomyces cerevisiae) [17,18,19]. The use of tall fescue biomass from saline soils is certainly important due to the EU Directive on Renewable Energy Sources 2018/2021 (RED II) defining the criteria for the sustainable development of biofuels, including biofuels from plant biomass, whose share in the transport sector by 2030 should amount to 3.5% [20]. Furthermore, the European Union has just completed the next stage of work on the new shape of the RES Directive (RED III), increasing the share of energy from RES to a minimum of 42.5% by 2030, including a 5.5% share of advanced biofuels for the transport sector.

In addition, in recent years, there has been an increasing emphasis on the need to sig-nificantly decrease the quantity of plastic waste (the Single-Use Plastics Directive) [21,22]. This is due to the fact that millions of tons of plastic waste (PE, PP, PET, PS, PVC) are produced every year around the world, from which products are obtained that are largely disposable and decompose over several hundred years. In order to meet the legal requirements and environmental expectations of responsible customers, the plastics industry is looking for environmentally friendly materials. In recent years, there has been growing interest in eco-friendly biodegradable materials. An example of an environmentally friendly plastic is polylactic acid (PLA), which is one of the leading biodegradable plastics in the market [23,24,25].

On the other hand, the use of cheap natural fillers from plant biomass to produce biocomposites based on still expensive biodegradable plastics may be a good way to improve the economics of their production [26]. In the field of natural fillers for the plastics industry, raw materials obtained from wood biomass (WPC composites) and fibrous plants (flax, hemp, jute, sisal, kenaf, etc.) have so far been the most popular [27,28]. The biomass of plants grown in degraded areas also seems to be potentially interesting, e.g., tall fescue biomass obtained from saline soils [13]. Materials consisting of biodegradable thermoplastic polymers with natural fillers can show functional properties like traditional polymers and can be used in industrial production, especially in the area of products with a short life cycle.

Currently, in the presented studies, we compared the use of tall fescue biomass for biorefining processes—bioethanol and fillers for polylactide biocomposites, derived from soils with different degrees of salinity.

2. Materials and Methods

2.1. Plant Material

The plant biomass was collected on 19 August 2021 from a two-year experimental cultivation of tall fescue (F. arundinacea cv. ‘Fawn’) in saline soil. Along the designated transect according to the NaCl concentration gradient, eight plots with an area of 1 m² were established. Then, the above-ground plant parts were carefully cut using a petrol lawn mower with a basket, and soil samples were collected for the salinity analysis. For the harvested plant material from individual plots, fresh and air-dry weight, moisture content, and the level of sodium ions were determined. In order to obtain the maximum biomass, the plant collection was carried out in the full growing season in the second half of August, and the transect area was excluded from the second swath made on the rest of the field on 15 July.

The obtained samples of F. arundinacea were dried at temperature of 30 °C and relative humidity of 40%. Moisture content in air-dry biomass were measured after drying at 105 °C in a HR73 Halogen Moisture Analyzer (Mettler Toledo, Greifensee, Switzerland).

2.2. Plant Cultivation

The field cultivation of F. arundinacea was located in Kościelec near Inowrocław (N 52°46′31.2″, E 18°09′18.6″; central Poland). The plantation with an area of approximately 850 m² was established in April 2020 from sowing seeds (15 kg∙ha⁻¹, a row spacing of 45 cm) on salt-degraded soil due to leakage of contaminated industrial water. In order to prepare the field for cultivation, in 2019, the disking, non-selective herbicide spraying and pre-winter plowing were performed. In the spring of the following year, the dragging and mineral fertilization (60 kg of N∙ha⁻¹, 80 kg of P₂O₅∙ha⁻¹ and 100 kg of K₂O∙ha⁻¹) were conducted. Then, pre-sowing tillage (two times harrow and cultivating unit), sowing seeds and rolling with a smooth roller were performed. To determine the biomass yield, F. arundinacea was cut by a drum mower, and the field-dried biomass was baled. In early spring of the second cultivation year, mineral fertilizer was sown (60 kg of N∙ha⁻¹, 80 kg of P₂O₅∙ha⁻¹ and 80 kg K₂O∙ha⁻¹). After the first and second harvesting, the amount of nitrogen in the soil was supplemented by 40 and 20 kg of N∙ha⁻¹, respectively.

2.3. Soil Salt Content

Soil for evaluating the salt content, weighing 750–1000 g, was collected from the root zone of plants growing in the experimental plots and dried to a constant weight. From a well-mixed sample, 20 mL of soil was collected and transferred to a glass beaker, into which twice as much distilled water (40 mL) was poured. The entire content of the beaker was thoroughly mixed, and the resulting soil solution was incubated at 20 °C for 3 h. Then, 30 min before the measurement, the solution was stirred vigorously for at least 10 s, and the solution was decanted from the above precipitate.

In the filtrate, salinity was measured using the conductivity method using the CPC-461 m (Elmetron, Poland) with the ECF-01 probe (2048/18), which enables measurement in immersion in a wide measuring range of 0–400 mS∙cm⁻¹. The obtained conductivity result was automatically converted using the CPC-461 m to the concentration in g of NaCl.

2.4. Sodium Content in F. arundinacea

The tall fescue biomass sample was ground using a mill, 0.5 g of mass was weighed with an accuracy of 0.0001 g and placed in a Teflon container for mineralization. A 5.0 mL volume of HNO₃ (65%) was added. The sample was subjected to microwave mineralization in a closed system in a mineralizer MAGNUM II (ERTEC, Wrocław, Poland) in three cycles: cycle 1–5 min, heating power 60%; cycle 2–5 min, heating power 80%; and cycle 3–10 min, heating power 100%. Then, the mineralized sample was transferred to a 50 mL flask and made up to the mark with demineralized water.

Moreover, on the basis of absorbance measurements of standard solutions, a standard curve was determined as a dependence of absorbance on concentration (0.5–2.0 μg∙mL⁻¹).

Test and standard sodium solutions were analyzed by atomic absorption spectrometry with acetylene–air flame atomization for reference samples at 589.0 nm on an AVANTA Σ atomic absorption spectrometer (GBC Scientific Equipment Ltd., Keysborough, VIC, Australia).

2.5. Bioethanol Production Process

2.5.1. Pretreatment of Tall Fescue Biomass

Tall fescue biomass was cut into 20–40 mm particles and then dried (50–55 °C) until constant weight was obtained. The material prepared in this way was ground using a knife mill (sieve 2 mm) SM-200 (Retsch, Hann, Germany).

In the next stage of pretreatment, the fescue biomass was treated with 1.5% sodium hydroxide for 5 h. The temperature of this process was 90 °C [29], and the NaOH/biomass weight ratio was 10:1. Then, for neutralization, the alkaline solution of the tested biomass was filtered using a Buchner funnel and washed and dried for 24 h at 50 °C.

2.5.2. Simultaneous Saccharification and Fermentation Process (SSF)

The SSF process was carried out in 100 mL Erlenmeyer flasks. The total volume of the treated tall fescue biomass was 40 mL, and the desired pH of 4.8 was maintained for the solution with 1 M sulfuric acid and 1 M sodium hydroxide. The enzymes, Flashzyme Plus 200 (AB Enzymes GmbH, Darmstadt, Germany) and Celluclast 1.5L (Merck Life Science Sp. z o. o., an affiliate of Merck KGaA, Darmstadt, Germany) (7:3 v/v), in the amount of 10 FPU∙g⁻¹ in total and non-hydrated lyophilized yeast (Saccharomyces cerevisiae) in the amount of 0.5 g∙L⁻¹ were also added to the tested biomass. The flasks (sealed with stoppers with fermentation tubes) were incubated at 36 °C on a shaker (200 rpm) for 72 h. All tests were performed in triplicate.

2.6. Biocomposites Production Process

2.6.1. Natural Fillers from Tall Fescue Biomass

Samples of dried tall fescue biomass were ground using a cutting mill SM 100 (Retsch, Haan, Germany), with a sieve separator with a mesh diameter of 1 mm. Then, a sieve analysis of the obtained natural fillers was performed on the Analysette 3 Spartan (Fritsch, Idar-Oberstein, Germany), and their humidity was determined on the MA.X2 moisture analyzer (Radwag, Radom, Poland).

2.6.2. Polymer Matrix

The biodegradable polylactide matrix (PLA) Ingeo 3251D (NatureWorks, Plymouth, MN, USA) designed for injection molding applications with a density of 1.24 g∙mL⁻¹ and a melt flow rate (MFR) of 80 g∙10 min⁻¹ (210 °C, 2.16 kg) was used.

2.6.3. Composites Preparation

Biocomposites containing 20 wt% fillers from F. arundinacea (FA) were compounded with PLA 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). Parameters of the compounding process: barrel temperature profile of 170–195 °C, extruder rotation speed of 110 rpm and throughput 10 kg∙h⁻¹.

The obtained biocomposite granules were dried to a humidity level below 0.02% in a Drywell DW12/10 molecular dehumidifier (Digicolor, Herford, Germany) at a temperature of 70 °C (dew point −40 °C).

Multipurpose test specimens for mechanical tests, i.e., type A in accordance with ISO 3167 [30], were molded by hydraulic injection molding machine Haitian Mars II MA600 (Haitian Plastics Machinery, Ningbo, China). Barrel temperature profile: 180 °C (hopper), 190 °C, 190 °C and 195 °C (nozzle). Mold temperature was set at 30 °C.

2.7. Analytical and Testing Methods

The chemical composition of tall fescue biomass before and after pretreatment was determined in accordance using standards, i.e., cellulose TAPPI T17 m-55 [31] and lignin TAPPI T13 m-54 [32], and the content of hemicellulose was the difference between holocellulose TAPPI T9 m- 54 [33] and cellulose.

A scanning electron microscope (SEM, S-3400N, Hitachi, Tokyo, Japan) under high-vacuum conditions was used to study the physical morphology of tall fescue biomass before and after chemical treatment. Test samples were covered with gold dust.

The concentration of ethanol formed during the SSF process was determined using liquid chromatography (Elite LaChrom by VWR-Hitachi), with the RI L-2490 detector, Rezex ROA 300 × 7.80 mm column (Phenomenex, Torrance, CA, USA), and the flow rate at 40 °C was 0.6 mL∙min⁻¹. The samples were loaded onto the column at 10 µL. The quantification was performed with the external standard method using the peak area (measurement and computer integration using the Ez-Chrom Elite software version 3.2.0).

Tensile and bending tests were carried out in accordance with the recommendations of ISO 527 [34,35] and ISO 178 [36], respectively, at room temperature using the Inspekt Table 50 universal testing machine (Hegewald & Peschke MPT, Nossen, Germany). In both tests, the crosshead speed was set to 5 mm∙min⁻¹. In addition, tensile modulus was carried out according to ISO 527-1, 2 using a MFA clip-on extensometer (MF Mess- & Feinwerktechnik, Velbert, Germany) with a nominal length of 50 mm.

2.8. Calculations

Ethanol yield (Y_s) from 100 g of tall fescue biomass dry matter (DM) was calculated according to equation [37]:

Y_s = (Et × 100)/M [g∙100 g⁻¹ of tall fescue biomass]

where Et—amount of ethanol in 1000 mL of tested sample [g], and M—mass of material weighed in 1000 mL fermentation sample [g].

In the next step, based on the ethanol yield from 100 g of tall fescue biomass, the amount of ethanol in L per ton of biomass dry matter [L∙Mg⁻¹] was calculated, and then the ethanol yield per hectare [m³∙ha⁻¹] was determined from the biomass yield.

2.9. Statistical Analysis

Ethanol fermentation of tall fescue biomass in triplicate was carried out, and the standard deviation was calculated using the ANOVA analysis of variance (Statistica 13.0 software, where p < 0.05).

3. Results and Discussion

3.1. Effect of Soil Salinity on Biomass Yield and Sodium Content in F. arundinacea

Our research provided novel solutions in the field of bioremediation of naturally or re-salinized agricultural land with the use of native facultative halophyte—Festuca arundinacea. In the first year of cultivation (2020) on saline soil, the yield of tall fescue hay was obtained at the level of 3.9 Mg∙ha⁻¹ (one cut), while in the following year, three harvests were performed (25 May, 15 July, and 22 September) with a total yield of 14.8 Mg∙ha⁻¹ (6.24, 4.29 and 4.24 Mg∙ha⁻¹, respectively). In Central Europe, the crop of F. arundinacea is usually harvested three times a year (May, July and September), but the total annual yield and individual cuts may vary significantly depending on the year of cultivation, weather conditions, soil abundance, fertilization level, etc. Field experiment conducted in Mydlniki near Cracow (Southern Poland) on non-saline soil in 2004–2006 showed an annual dry matter yield of tall fescue (three cuts) of about 8–10 Mg∙ha⁻¹ [38]. A high yield of tall fescue was obtained in the cultivation located on the organic soil in the Randow river valley (Northeast Germany). The meadow sward was dominated by F. arundinacea ‘Fawn’, and its share was 100% in the first and second year of cultivation and 99% in the fourth year. In the first year of cultivation (after seed sowing at first decade of September), three cuts were harvested, while in the second and fourth year—4 and 5 cuts, respectively. In these years, total annual yield of F. arundinacea dry matter was 12.97, 14.40 and 17.43 Mg∙ha⁻¹ [39]. In the conditions of the Czech Republic, dry matter yield of a mixture of Lolium perenne and F. arundinacea (first cut in June) ranged from 5.3 to 12.7 Mg∙ha⁻¹ depending on growing season as well as the sowing dates (June, September or October) and seed rate (10 or 20 kg∙ha⁻¹) [40].

In our investigations, in the second year of field cultivation (2021), tall fescue formed dense turf without weeds (Figure 1).

The yield of above-ground plant parts harvested from the plots along the soil salinity gradient varied within a wide range from 1.75 to 2.90 kg∙m⁻² and from 527.85 to 902.48 g∙m⁻² for fresh and air-dry biomass, respectively. Biomass loss after air-drying averaged 70.64%, falling within a relatively narrow spread of 68.73 to 74.09%. In turn, the average moisture content in the air-dry biomass of tall fescue reached 10.56% (

Table 1. Biomass yield of F. arundinacea on experimental plots in the field cultivation.

Plots	Fresh Biomass Yield (kg∙m−2)	Biomass Loss after Air-Drying (%)	Air-Dry Biomass Yield (g∙m−2)	Dry Biomass Yield (g∙m−2)	Moisture Content in Fresh Biomass (%)	Moisture Content in Air-Dry Biomass (%)
FA_1	2.05	68.73	641.10	578.37	71.79	9.79
FA_2	1.75	69.84	527.85	474.11	72.91	10.18
FA_3	2.05	70.56	603.53	541.16	73.60	10.34
FA_4	2.75	74.09	712.50	636.76	76.85	10.63
FA_5	2.25	71.20	648.08	576.76	74.37	11.01
FA_6	2.55	71.15	735.57	655.43	74.30	10.90
FA_7	2.55	70.71	746.95	663.96	73.96	11.11
FA_8	2.90	68.88	902.48	807.67	72.15	10.51
Mean ± SD	2.36 ± 0.37	70.64 ± 1.58	689.76 ± 105.41	616.78 ± 93.35	73.74 ± 1.48	10.56 ± 0.42
V [%]	15.68	2.23	15.28	15.13	2.00	3.99

SD—standard deviation, V—variability coefficient (n = 8 study plots).

). The approximate air-dry biomass yield estimated on this basis was 6.9 Mg∙ha⁻¹.

On experimental plots in the field cultivation of F. arundinacea, the concentration of salts in plants and soil was determined (

Table 2. Salt concentration in plants and soil on experimental plots of tall fescue.

Plots	Na+ Content in Dry Biomass (mg∙g−1)	Soil Salinity (g NaCl∙L−1)
FA_1	2.72 ± 0.30	6.85 ± 0.18
FA_2	2.45 ± 0.11	6.24 ± 0.09
FA_3	7.17 ± 1.18	5.76 ± 0.38
FA_4	6.45 ± 0.27	4.75 ± 0.21
FA_5	8.38 ± 0.21	4.02 ± 0.15
FA_6	7.71 ± 0.73	2.82 ± 0.25
FA_7	8.57 ± 0.48	1.49 ± 0.06
FA_8	8.66 ± 0.88	0.70 ± 0.06
Mean ± SD	6.51 ± 2.37	4.08 ± 2.10
V [%]	36.44	51.55

SD—standard deviation, V—variability coefficient (n = 8 study plots).

).

The average content of sodium ions in the above-ground plant parts of tall fescue was high and amounted to 6.51 mg∙g⁻¹ of dry matter. However, in individual experimental plots, it ranged widely from 2.45 to 8.66 mg∙g⁻¹ of dry matter. Similarly, the soil salinity (expressed as sodium chloride equivalent) remained high, though variable, from 0.70 to 6.85 g∙L⁻¹ with a variability coefficient of 51.55%.

Our research showed a strong negative correlation between soil salinity and air-dry biomass yield of F. arundinacea. At the lowest content of NaCl in the soil (0.70 g∙L⁻¹), tall fescue yield was 902.48 g∙m⁻², while at the highest soil salinity (more than 5.50 g∙L⁻¹), it decreased to 527.85–641.10 g∙m⁻² (Figure 2).

The negative effect of salinity on tall fescue yield has been shown by two field experiments conducted in Turkey. The average dry matter yield from three consecutive years of F. arundinacea cultivation at one cut was 5.94 and 4.59 Mg∙ha⁻¹ for non-saline and highly saline soil, respectively [41]. In the second experiment, a gradual decrease in the yield of tall fescue was obtained during irrigation with water having an increased salt content [42]. Similar observations were obtained for experiments in pots. Dry weight yield of F. arundinacea ‘Fawn’ for the first cut reached 41.08, 28.64, 13.08 and 11.96 g∙m⁻² for non-saline control and irrigation of water with 4000, 8000 and 12,000 ppm of NaCl, respectively [43].

It was interesting that in our field experiment a relatively strong negative correlation was found between the salt content in the soil and above-ground plant parts of F. arundinacea (Figure 3).

However, a detailed analysis of this graph shows that the sodium content in plants changes abruptly, staying at a similar level in quite a wide range of soil salinity: 0.70–1.49, 2.82–5.76 and 6.24–6.85 g NaCl∙L⁻¹. It confirms the results of the pot experiments with irrigation using saline water [43]. At the lowest level of water salinity, the sodium content in plants of F. arundinacea ‘Fawn’ clearly increased compared to the non-saline control, while at higher concentrations of NaCl, it strongly decreased, remaining at a similar range. In addition, in these studies, an inverse relationship was found for potassium ions in tall fescue.

3.2. Bioethanol Production Process

Tall fescue biomass obtained from saline sites (average sample) before and after alkaline treatment was analyzed for chemical composition (cellulose, hemicellulose, lignin) [13]. The biomass was ground by a knife mill with 2 mm sieve and treated with alkali, i.e., 1.5% NaOH. The chemical structure analysis of tall fescue biomass showed that the alkaline treatment effect caused an increase in the content of cellulose from 33.69% to 50.41% (approx. 17%), a potential substrate in the process of obtaining biofuels. Moreover, partial degradation of hemicellulose from 34.74% to 25.23% (over 9%) was found. However, in the case of lignin content, which is a strong obstacle in the biomass conversion process, a decrease from 17.08% to 12.35% (almost 5%) was observed after alkaline treatment. Overall, the biomass of tall fescue from saline areas exhibits delignification after alkaline treatment and additionally solubilization of part of hemicellulose [13,44].

The effect of alkaline treatment on tall fescue biomass from saline soils was confirmed using Scanning Electron Microscopy (Hitachi, Tokyo, Japan) shown in Figure 4.

Significant changes on the surface of tall fescue biomass were observed before and after pretreatment. Figure 4a shows that untreated biomass has intact and rigid structure, which effectively blocks access to lignocellulose. On the other hand, after pretreatment with sodium hydroxide (Figure 4b), SEM image of biomass shows structural damage and partial purification of the biomass surface. It can be concluded that NaOH degrades the bonds in lignocellulose, which increases its surface area and makes it more accessible to enzymes [45,46,47].

Then, the SSF process was performed, which ensures optimal synergy of enzymes and distiller’s microorganisms. The enzymes Flashzyme Plus 200 (AB Enzymes GmbH, Darmstadt, Germany) and Celluclast 1.5 L (7:3 v/v) in the amount of 10 FPU∙g⁻¹ in total and the yeast Saccharomyces cerevisiae in the amount of 0.5 g∙L⁻¹ were used. Fermentation tests were performed on tall fescue biomass samples obtained from saline soils with different levels of salinity, and the amount of ethanol was determined using High Performance Liquid Chromatography (Figure 5).

In the SSF process, ethanol was obtained in the range of 8.62–10.60 g∙L⁻¹ for all tall fescue biomass samples obtained from saline soils in the range of 0.70–6.85 g NaCl∙L⁻¹, and for which the Na⁺ content in dry matter was from 2.45 to 8.66 mg∙g⁻¹. It was found that both the level of soil salinity and sodium content in tall fescue biomass had no significant effect on the amount of ethanol obtained. For example, for sample FA_1 (soil salinity: 6.85 g NaCl∙L⁻¹ and Na⁺ content in biomass: 2.72 mg∙g⁻¹) and sample FA_7 (soil salinity: 1.49 g NaCl∙L⁻¹ and Na⁺ content in biomass: 8.57 mg∙g⁻¹), despite the extreme values of soil salinity and sodium content in the biomass, the concentration of ethanol was obtained at a similar level of approx. 9.7 g∙L⁻¹. In conclusion, in general, tall fescue biomass from saline soils has great potential of producing bioethanol with an average concentration of 9.66 g∙L⁻¹ (19.32 g∙100 g⁻¹ biomass), which makes obtaining approx. 245 L of ethanol per ton of raw material (DM) possible and from one hectare of degraded soil approx. 1.7 m³ of ethanol.

It is worth mentioning that in literature reports, tall fescue is also mentioned as a crop that is a potential raw material for the production of second-generation bioethanol [48]. Research on obtaining bioethanol from tall fescue biomass was carried out, among others, by Kumar and Murthy, who obtained 360 L·Mg⁻¹ of ethanol after chemical pretreatment, followed by enzymatic hydrolysis and fermentation. They tested a total of three different grasses, including perennial ryegrass and bentgrass, but the highest ethanol yield was obtained from tall fescue [46]. Horita and Kiyoshi also conducted research on the yield of lignocellulosic ethanol from tall fescue biomass. They compared ethanol yields from several plant raw materials, including sorghum and bagasse. It turned out that after the SSF process, the yield of ethanol for bagasse was about 70 mg·g⁻¹ DM, for tall fescue was about 90 mg·g⁻¹ DM and sorghum was about 120 mg·g⁻¹ DM [49].

3.3. Biocomposites Production Process

Research was also carried out on the possibility of using tall fescue biomass obtained from areas with different salinity levels in the plastics processing industry, developing biodegradable polymer composites in which some plastics were replaced with natural fillers. Natural fillers were prepared from tall fescue biomass with particles of less than 1 mm. Moisture analysis and sieve analysis of the obtained natural fillers were performed. The detailed share of the individual fractions is presented in

Table 3. Particle size distribution and humidity of fillers from F. arundinacea biomass.

Plant Biomass	Humidity (%)	Particle Size Distribution (%)
		1 mm	0.5 mm	0.4 mm	0.25 mm	0.2 mm	0.1 mm	Below 0.1 mm
FA_1	6.73	1.2	8.3	48.3	30.5	2.5	2.7	6.5
FA_2	7.65	0.8	3.5	51.3	29.3	4.7	3.2	7.2
FA_3	6.57	1.1	2.8	50.6	31.2	3.7	2.8	7.8
FA_4	6.33	0.8	3.1	49.5	30.6	4.2	2.9	8.9
FA_5	7.25	1.2	2.6	51.2	32.3	2.5	3.2	7.0
FA_6	6.84	1.1	3.6	40.9	40.2	3.4	3.6	7.2
FA_7	6.20	1.7	1.6	52.3	33.9	2.1	2.6	5.8
FA_8	6.54	1.5	2.1	49.2	35.4	2.1	2.4	7.3

.

The bulk density of tall fescue fillers (average 0.215 kg∙L⁻¹) was also determined.

The effect of various tall fescue fillers on the tensile and flexural properties of PLA composites are shown in

Table 4. Tensile and flexural properties of PLA/tall fescue composites.

Sample	Tensile Strength δM (MPa)	Tensile Modulus Et (GPa)	Flexural Strength δfM (MPa)	Flexural Modulus Ef (GPa)
PLA 3251D	62.1 ± 0.5	2.34 ± 0.07	108.1 ± 0.4	2.21 ± 0.21
PLA-FA_1	47.2 ± 0.6	3.00 ± 0.05	83.8 ± 0.5	2.80 ± 0.14
PLA-FA_2	49.3 ± 0.9	3.14 ± 0.09	88.2 ± 0.9	2.92 ± 0.20
PLA-FA_3	46.6 ± 1.1	2.97 ± 0.10	82.6 ± 0.8	2.77 ± 0.17
PLA-FA_4	47.8 ± 0.9	3.06 ± 0.08	84.6 ± 0.8	2.85 ± 0.22
PLA-FA_5	46.1 ± 0.7	2.93 ± 0.07	82.2 ± 0.8	2.74 ± 0.09
PLA-FA_6	44.7 ± 0.6	2.85 ± 0.06	79.5 ± 1.1	2.66 ± 0.11
PLA-FA_7	46.8 ± 0.9	2.98 ± 0.11	83.5 ± 1.0	2.79 ± 0.09
PLA-FA_8	48.0 ± 0.6	3.06 ± 0.09	84.9 ± 0.9	2.83 ± 0.10

.

The use of 20% by weight of F. arundinacea biomass fillers in PLA-based composites reduced tensile strength by an average of 24%. However, it can be observed that these changes depend on the type of biomass used and range from 21% for sample FA_2 (soil salinity: 6.24 g NaCl∙L⁻¹ and Na⁺ content in biomass: 2.45 mg∙g⁻¹) to 28% for sample FA_6 (soil salinity: 2.82 g NaCl∙L⁻¹ and Na⁺ content in biomass: 7.71 mg∙g⁻¹). The use of tall fescue biomass natural fillers increased the modulus of elasticity of composites in relation to pure PLA—by an average of 28%. The best results were obtained for the FA_2 filler (about 34%), and the smallest increase in the module was recorded for the FA_6 filler (about 22%). The flexural strength of the composites at 20% by weight of the filler content decreased by about 22%. The modulus of elasticity determined during bending increased by an average of 26% compared to pure PLA.

It was found that PLA composites with fillers from tall fescue biomass did not show effective interfacial adhesion [50]. The lack of full adhesion at the interface of individual components may result from the chemical composition of lignocellulosic biomass [51,52]. In SEM images, this phenomenon is visible in the form of a gap between the polymer matrix and the filler (Figure 6).

The interfacial bonding forces of natural fillers with the polymer matrix were low, and even when low force was acting on the samples, the fillers detached from the matrix [53]. After exceeding this value, all the force acted only on the polymer matrix of the composite sample [54]. In addition, the value of the decrease in tensile and bending strength can be correlated with the type of reed fescue biomass used. It was observed that the FA_2 filler has the smallest effect on reducing the tensile and flexural strength of composites and the most favorable effect on increasing the modulus of elasticity. It should be added that this filler, despite being obtained from a crop with one of the highest salinities, was characterized using the lowest Na⁺ content in biomass.

4. Conclusions

• Festuca arundinacea was cultivated on soil with a high concentration of NaCl in the top layer (up to 6 g∙L⁻¹), and in the second year of cultivation, it formed a dense sod, produced three cuts and a medium high yield (14.8 Mg∙ha⁻¹).
• In this work, the tall fescue biomass obtained from the soil with different salinities was used in the process of obtaining bioethanol and green composites based on the biodegradable polymer PLA.
• It was found that the content of Na⁺ in tall fescue biomass had no significant effect on the amount of ethanol obtained (average: 19.32 g∙100 g⁻¹ of raw material), which may result from leaching of sodium ions during alkaline pretreatment.
• The possibility of using tall fescue biomass with the lowest Na⁺ content in biomass (approx. 2.5 mg∙g⁻¹) from an area with high salinity (above 6 g NaCl∙L⁻¹) as fillers for composites based on PLA was demonstrated. Moreover, for this biomass, the highest value of bioethanol concentration was obtained, i.e., 10.6 g∙L⁻¹ (21.2 g∙100 g⁻¹ of raw material).
• In summary, F. arundinacea can be successfully used for bioremediation of saline habitats and its biomass for industrial applications such as advanced biofuels and as natural fillers to improve the economics of using biodegradable polymers in composites.