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Article

Green Valorization of Alfalfa into Sustainable Lignocellulosic Films for Packaging Applications

by
Sandeep Paudel
and
Srinivas Janaswamy
*
Department of Dairy and Food Science, South Dakota State University, Brookings, SD 57007, USA
*
Author to whom correspondence should be addressed.
Appl. Sci. 2025, 15(22), 11889; https://doi.org/10.3390/app152211889 (registering DOI)
Submission received: 24 October 2025 / Revised: 2 November 2025 / Accepted: 7 November 2025 / Published: 8 November 2025
(This article belongs to the Special Issue Sustainability in Functional Textiles: Materials, Technology and Design)
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Abstract

Plastic-based materials dominate the packaging industry. However, their non-biodegradability has increased the need for sustainable alternatives. Biopolymers, mainly lignocellulose from agricultural residues, offer renewable, eco-friendly options in this context. This study reports the development of lignocellulosic films from alfalfa (Medicago sativa) through green valorization of its biomass. Alfalfa lignocellulosic extract (ALE) was extracted using 50% NaOH, solubilized in 68% ZnCl2, crosslinked with CaCl2, and plasticized with sorbitol. The concentrations of ALE, CaCl2, and sorbitol were optimized using the Box–Behnken Design, focusing on increasing tensile strength (TS), elongation at break (EB), and reducing water vapor permeability (WVP) of the films. The optimized film formulation (0.5 g ALE, 453.8 mM CaCl2, 1.5% sorbitol) showed a TS of 11.2 ± 0.7 MPa, EB of 5.8 ± 0.9%, and WVP of 1.2 ± 0.2 × 10−10 g m−1 s−1 Pa−1. The film effectively blocked UV–Vis–IR light and exhibited notable antioxidant activity, making it suitable for packaging light-sensitive and oxidation-sensitive foods. Additionally, it achieved over 90% biodegradation within 29 days under 24% soil moisture. These findings demonstrate a sustainable approach to upcycling agricultural residues into functional products, offering a practical alternative to traditional plastics and supporting a circular bioeconomy, while adding value for alfalfa producers.

1. Introduction

The invention of plastic in 1907 revolutionized the packaging industry. Before this breakthrough, traditional packaging materials such as leaves, animal skins, and wooden barrels were gradually replaced during the Industrial Revolution by metal cans, paper bags, and glass containers. However, the large-scale production of plastics, driven by their durability, corrosion resistance, light weight, mechanical strength, and affordability, quickly led to their dominance across various industries [1,2,3]. Consequently, modern society has entered a “plastic era,” with plastics becoming indispensable in packaging, construction, automotive, and healthcare applications. As of 2023, global plastic production has surpassed 11.5 billion metric tons [4], with about 40% used in packaging and plastics comprising nearly 45% of all packaging materials [1,5]. Despite their versatility, the non-biodegradable nature of plastics has resulted in severe environmental challenges. Plastic waste can persist for centuries, and approximately 91% of discarded plastics are either incinerated, releasing greenhouse gases such as CO2 and CO, or accumulate in landfills and oceans, damaging terrestrial and marine ecosystems [5,6,7]. Over time, these materials degrade into microplastics and nanoplastics, posing serious risks to both environmental and human health [8,9,10]. Each year, an estimated 100,000 marine animals perish from plastic ingestion and entanglement [11,12,13]. Furthermore, human exposure to microplastics is estimated at 269 mg per person per day [14], with particles detected in the brain, blood, lungs, placenta, semen, and breast milk [15,16,17,18,19,20,21,22]. Such exposure has been linked to adverse reproductive and neurological effects [23,24,25].
To mitigate these escalating environmental and health concerns, the development of sustainable, biodegradable, and eco-friendly packaging materials has become a global priority. Biopolymers such as cellulose, chitosan, starch, and proteins have emerged as promising alternatives to petroleum-based plastics. Among these, cellulose stands out as the most abundant, renewable, biocompatible, and mechanically robust biopolymer [26,27,28]. For a truly sustainable approach, agricultural residues and biomass represent ideal cellulose sources [1,29,30]. In this context, alfalfa (Medicago sativa), often referred to as the “queen of forage,” offers significant potential as a lignocellulosic feedstock. Alfalfa is a perennial legume widely cultivated for its high nutritional value in animal feed and its ability to fix nitrogen, thereby enhancing soil fertility [31,32]. Major producers include the United States, Russia, and Argentina, with global production reaching approximately 284.56 million metric tons annually [33,34]. Field residues account for nearly 25% of total alfalfa biomass [35], which amounts to approximately 71.14 million metric tons. With such substantial biomass production, alfalfa is a sustainable option for the development of biodegradable films. Additionally, the lignocellulosic content of around 27% [36] enhances its value. This biomass is also rich in calcium (21 mg/g) [36,37], which can help develop robust films and make alfalfa a suitable resource. Assuming half of these residues remain in the field to maintain soil health, the remainder could yield approximately 8.35 million metric tons of alfalfa lignocellulosic extract (ALE). This quantity could produce roughly 167 billion square meters of biodegradable film, potentially replacing a comparable volume of plastic packaging. Economically, this valorization could generate an estimated annual value of USD 5.34 billion, assuming a premium of USD 150 per ton of alfalfa biomass [38].
This study investigates the green valorization of alfalfa field residues through alkali treatment using 50% NaOH to extract lignocellulosic materials for the fabrication of biodegradable packaging films. Lignin is known to enhance the UV-blocking, antioxidant, antimicrobial, hydrophobic, and mechanical properties of cellulose films [28,39,40,41,42]. The extracted lignocellulosic residue retains the naturally occurring lignin from the alfalfa biomass, which improves the film properties compared to those of the alfalfa cellulose film. The films were formulated and optimized to evaluate their structural, mechanical, and functional properties. The results demonstrate a novel and sustainable pathway to transform agricultural residues into high-value, eco-friendly packaging materials, supporting both environmental protection and agricultural profitability.

2. Materials and Methods

2.1. Materials

Alfalfa field residue was collected from Watertown, South Dakota. The chemicals sodium hydroxide, zinc chloride, calcium chloride dihydrate, sorbitol, calcium sulfate, and potassium sulfate were purchased from VWR International, Radnor, PA, USA. Ethanol was obtained from the Department of Physics, Chemistry, and Biochemistry at South Dakota State University.

2.2. Extraction of Lignocellulosic Residue, Film Preparation, Film Optimization, and Characterization

The extraction of lignocellulosic residue, film preparation, film optimization, and characterization were performed following the established protocols [43,44]. Briefly, the dried alfalfa was ground to a 60-mesh powder and treated with a 50% (w/v) NaOH solution. The mixture was stirred at 300 rpm and maintained at 35 °C for 8 h. The treated mixture was washed thoroughly with distilled water through a 100-mesh sieve until the pH was neutral. The resulting alfalfa lignocellulosic extract (ALE) was oven-dried at 40 °C for 12 h, yielding 23.5 ± 1.0%. It was ground to a 60-mesh powder and stored in airtight bags for subsequent use. The obtained yield was comparable to previously reported yields of 24.4% [44] and 27.4% [36].
For film preparation, the ALE powder was swelled at 83 °C for 2 h, dissolved in 68% ZnCl2 solution for 30 min, crosslinked with CaCl2 for 10 min, and plasticized with sorbitol for 5 min. The film-forming solution was cast onto a glass plate using a handheld applicator with a 1 mm clearance, and the films were regenerated using ethanol, framed, washed with distilled water, and dried to obtain uniform films. Optimization of the film formulation was performed using a Box–Behnken Design (BBD) to maximize tensile strength (TS) and elongation at break (EB) while minimizing water vapor permeability (WVP). The independent variables were ALE content (0.3–0.5 g), CaCl2 concentration (200–500 mM), and sorbitol content (0.5–1.5%). Fifteen experimental combinations were generated within these ranges. The resulting films had thicknesses ranging from 0.04 mm to 0.08 mm.
The optimized film was evaluated for TS, EB, and WVP, along with its color, transparency, electromagnetic transmittance, absorption coefficient, Fourier-transform infrared spectroscopy (FTIR), antioxidant activity, moisture content, water solubility, water contact angle, water absorption and kinetics, and soil biodegradation behavior [43]. Tensile strength and elongation at break of the films were determined using a Texture Analyzer (Stable Micro Systems, TA-HD Plus, Serial No. 5529, Surrey, UK). The water vapor permeability (WVP) was evaluated by sealing a jar containing anhydrous CaSO4 (to maintain 0% relative humidity, RH) with the film sample. It was placed inside a desiccator regulated at approximately 97% RH using a saturated K2SO4 solution. The weight was recorded periodically for 8 h, and films’ WVP were calculated. The color attributes were analyzed using a Nix Pro 2 color sensor (Model no: NIXPRO002, Nix Sensor Ltd., Hamilton, ON, Canada) on the Hunter scale. Transparency was examined using a UV–Visible spectrophotometer (Model UV-1600PC, 10037-436, VWR International, Radnor, PA, USA) by recording transmittance at 600 nm, while the overall UV–Vis–IR region spectral transmittance was measured within the range of 190–1060 nm. FTIR spectra were collected between 400 and 4000 cm−1 with 36 scans at a resolution of 4 cm−1 to identify functional groups. Antioxidant activity was performed by the DPPH radical scavenging method, and IC50 values were calculated. Film moisture content, water solubility, water absorption, and biodegradability were determined using a gravimetric method. For moisture determination, films were oven-dried at 110 °C for 24 h. For water solubility, pre-dried films were immersed in water and agitated at 175 rpm for 24 h, followed by drying for an additional 24 h. The water contact angle was determined using a Dropometer (Droplet Lab, Markham, ON, Canada) by placing a water droplet on the film surface and recording the angle immediately. Water absorption was measured by immersing the pre-dried films in distilled water, with weight taken at 5, 10, 15, 30, 60, 90, and 120 min. The absorption kinetics were analyzed using 9 established mathematical models (Peleg, Singh, Gornicki, Pilosof, Czel and Czigany, Peppas, Vega-Gálvez, García-Pascual, and Weibull). For biodegradation, the films were buried in soil (at 24% moisture), and their remaining mass was weighed every other day until approximately 90% degradation was achieved. Biodegradation kinetics were fitted to first- and second-order reaction models. The equations used for calculations are provided in the Supplementary File S1. The lab temperature was 22 ± 2 °C with RH of 47 ± 2% throughout the experiment.

2.3. Statistical Analysis

Experimental design and optimization were conducted using State-Ease 360 software (Version 23.1.4). Statistical analyses were performed using RStudio (Version 2024.09.1+394) and Microsoft Excel for Mac (Version 16.88 (24081116)) with Solver Add-ins. The actual and predicted values for TS, EB, and WVP were compared using Welch’s two-sample t-test at a 95% confidence level. Water absorption and soil biodegradation kinetics were analyzed using nonlinear regression models.

3. Results and Discussion

3.1. Film Optimization

Fifteen film formulations with different amounts of ALE, CaCl2, and sorbitol were prepared and characterized for tensile strength (TS), elongation at break (EB), and water vapor permeability (WVP). The films showed TS, EB, and WVP values ranging from 2.2 ± 0.7 to 12.6 ± 0.3 MPa, 2.9 ± 0.7 to 9.1 ± 0.1%, and 0.9 ± 0.1 to 1.6 ± 0.1 × 10−10 g m−1 s−1 Pa−1, respectively (Table 1). The influence of each independent variable on the film responses is displayed in the 3D contour plots (Figure 1). Increasing the concentrations of ALE, CaCl2, and sorbitol significantly improved TS (Figure 1a–c), slightly decreased EB (Figure 1d–f), and markedly reduced WVP, except for a subtle increase associated with sorbitol (Figure 1g–i). A synergistic effect among the variables was also observed. The combination of ALE and CaCl2 showed the most significant improvement in TS; for example, TS increased from 2.4 ± 0.4 MPa (ALE1) to 12.6 ± 0.3 MPa (ALE4) as ALE and CaCl2 increased from 0.3 g and 200 mM to 0.5 g and 500 mM, respectively. Similar positive interactions were observed between ALE–sorbitol and CaCl2–sorbitol pairs. However, these same combinations resulted in decreased EB values (6.7 ± 0.9% to 2.9 ± 0.7%), likely due to the formation of a denser network. WVP also reduced from 1.6 ± 0.1 to 0.9 ± 0.1 × 10−10 g m−1 s−1 Pa−1, confirming improved barrier properties.
Once ZnCl2 solution is added to the lignocellulosic residue, O3H⋯Zn bonds form by breaking the rigid intra-chain O3⋯O5H bonds, making the lignocellulosic chains more flexible and resulting in a hydrogel. The loosened chains then allow Ca2+ ions to crosslink and reinforce the Zn-lignocellulose network through ionic crosslinking between hydroxyl groups, creating a stronger lignocellulosic network and increasing the tensile strength (TS) of the films [45,46,47]. However, such reinforcement decreases cellulose chain mobility and pore volume, thereby reducing both EB and WVP. Likewise, ALE enrichment increases matrix density, while sorbitol acts as a mild plasticizer, preserving some flexibility. Similar effects have been observed in films made from cellulose [46], corncob [48], soyhulls [43,49,50], and alfalfa cellulosic residue [44].
From the Box–Behnken Design analysis, the responses TS, EB, and WVP were well fitted with the quadratic model fittings, with R2 values of 0.9343–0.9670, p-values for models <0.05, and p-values for lack of fit >0.05 (Table 2). These values support the model fitting (Equations (1)–(3)). The predicted optimal formulation—0.5 g ALE, 453.8 mM CaCl2, and 1.5% sorbitol—was estimated to yield a TS of 11.4 MPa, EB of 5.5%, and WVP of 1.0 × 10−10 g m−1 s−1 Pa−1. The experimental results (11.2 ± 0.7 MPa, 5.8 ± 0.9%, and 1.2 ± 0.2 × 10−10 g m−1 s−1 Pa−1; p > 0.05) closely matched these predictions. Their respective p-values were 0.7103, 0.7387, and 0.4773, further validating the model fitting at a 95% confidence level. Some of the comparable biopolymer films’ TS, EB, and WVP are listed in Table 3.
Table 2. ANOVA of quadratic model fitting for the responses tensile strength (TS), elongation at break (EB, and water vapor permeability (WVP).
Table 2. ANOVA of quadratic model fitting for the responses tensile strength (TS), elongation at break (EB, and water vapor permeability (WVP).
ResponseSourceF-Valuep-ValueR2
TSModel16.290.00340.9670
Lack of fit7.520.1197
EBModel10.410.00950.9493
Lack of fit1.020.5295
WVPModel7.910.01740.9343
Lack of fit2.530.2957
TS = 8.20 + 3.12A + 2.49B − 0.07C + 1.17AB − 0.09AC − 0.09BC − 1.28A2 − 0.58B2 − 0.70C2
EB = 5.91 − 1.03A − 0.93B − 0.43C − 0.35AB + 1.32AC + 0.29BC − 0.18A2 − 0.54B2 + 0.83C2
WVP = 1.19 − 0.10A − 0.17B + 0.03C − 0.02AB − 0.01AC − 0.01BC + 0.05A2 − 0.01B2 − 0.01C2
Wherein, A: Alfalfa lignocellulose extract (ALE, g), B: CaCl2 (mM), and C: Sorbitol (%), and the values will be in the range of −1 to 1, representing lower to higher values of respective independent variables.
Table 3. A comparison of tensile strength (TS, MPa), elongation at break (EB, %), and water vapor permeability (WVP, 10−10 g m−1 s−1 Pa−1) of a few biodegradable films.
Table 3. A comparison of tensile strength (TS, MPa), elongation at break (EB, %), and water vapor permeability (WVP, 10−10 g m−1 s−1 Pa−1) of a few biodegradable films.
SampleTSEBWVPReference
Alfalfa lignocellulose11.2 ± 0.75.8 ± 0.91.2 ± 0.2This Study
Alfalfa cellulose16.910.10.47[44]
Avocado peel fiber7.2–15.75.2–13.62.4–2.5[51]
Banana peel fiber16.3–31.34.9–13.02.4–3.6 × 103[52]
Carboxymethyl cellulose2.1–22.06.7–45.148.6–214.0[53]
Cellulose0.3–22.44.3–13.20.6–12.2[46]
Cellulose acetate 0.1–3.20.2–9.547.2–233.3[54]
Corncob cellulose4.715.41.8[48]
Cow dung cellulose2.2–4.29.7–11.01.0–1.5[55]
Grapevine cellulose15.4–18.26.1–8.60.7–1.4[56]
Palm sprout peel4.0–11.24.8–80.40.1–9.2 × 105[57]
Soyhull cellulose6.330.20.9[49]
Soyhull lignocellulose 9.38.80.3[43]
Soyhull lignocellulose extract16.814.70.2[50]
Spent coffee grounds lignocellulose8.4–26.83.8–7.90.8–1.8[58]
Switchgrass lignocellulose9.9–14.73.4–4.70.1–0.2[59]
Switchgrass lignocellulosic extract 8.9–12.72.2–2.40.2–0.3[60]
Wheat straw fiber5.3–6.616.4–27.31.9–2.4[61]

3.2. Optimized Film Characterization

3.2.1. Optical Properties

The optical properties of packaging are crucial to consumer perception, as color can greatly influence purchasing decisions. The film exhibited Hunter’s color values of L* = 57.8 ± 1.0, a* = 9.6 ± 0.3, and b* = 24.7 ± 0.9. The L* value, slightly above 50, along with small positive a* and b* values, suggests that the film is slightly tilted toward whiteness with subtle red and yellow hues. The calculated whiteness index (WI), yellowness index (YI), and total color difference (TCD) were 49.9 ± 0.5, 61.0 ± 1.0, and 41.5 ± 0.4, respectively. These values indicate that the film has a neutral gray base with a strong yellow tint, making it appear slightly darker. The presence of chromophore functional groups in lignin has contributed to the darker color of the film [62]. The moderate brightness and whiteness of the ALE film reflect natural, sustainable materials and enhance its visual appeal, potentially boosting market preference. In comparison, the color values are similar to those reported for spent coffee grounds films [58], but lower than switchgrass [60] and soyhull-derived lignocellulose films [43,50].
The film was translucent, exhibiting a transparency of 20.0 ± 0.7% mm−1. This value is comparable to that of low-density polyethylene (LDPE) films, which typically show 15–20% transparency [63], indicating it is suitable for uses where optical clarity is desirable. The film also demonstrated excellent light-blocking properties across the UV–Vis–IR spectrum (Figure 2a). In the UVC region, the light transmittance at 190 nm was 32.3 ± 1.3%, gradually increasing to 52.0 ± 1.3% at 260 nm. Thereafter, in the UVB region, transmittance steadily decreased to 1.4 ± 0.3% at 310 nm, followed by a slight increase in the UVA region, reaching 4.2 ± 0.7% at 390 nm. Transmittance slightly increased in the visible and infrared regions, reaching 11.6 ± 1.0% at 700 nm and 14.5 ± 1.5% at 1060 nm, respectively. The film’s UV-blocking properties are helpful in packaging light-sensitive products, as they help preserve vitamins, lipids, and pigments, thereby maintaining the nutritional and aesthetic quality of food [64]. Notably, the light transmittance of the ALE film was lower than that of the alfalfa cellulose film [44], likely because the residual lignin fraction contains functional chromophore groups in the lignocellulosic extract, which increases UV absorption and light shielding [62,65].
Conversely, the absorbance coefficient decreased in the UVC region, from 22.6 ± 0.8 at 190 nm to 13.1 ± 0.5 at 260 nm (Figure 2a). It then increased to 87.9 ± 1.5 at 310 nm. In the UVA region, it decreased to 61.7 ± 0.3 at 390 nm, followed by further reductions in the visible and infrared regions, to 43.1 ± 1.6 at 700 nm and 38.6 ± 2.0 at 1060 nm, respectively. The relatively high absorbance, especially in the UV region, indicates an enhanced light-absorbing capacity, likely due to residual lignin in the lignocellulosic extract.
FTIR analysis indicates that ALE is a heterogeneous mixture of cellulose, xylan, mannan, and lignin, which are retained within the film matrix. In the film, the characteristic peaks of ALE were either retained, shifted, disappeared, or newly formed (Figure 2b,c), likely due to chemical interactions with ZnCl2, CaCl2, and sorbitol. The retained peaks appeared at 1155, 1265, 1330, and 3362 cm−1, whereas the shifted peaks occurred at 808 → 807, 895 → 896, 1020 → 1014, 1227 → 1234, 1367 → 1369, 1418 → 1419, 1454 → 1448, 1508 → 1506, 1642 → 1641, 2892 → 2898, 2919 → 2920, 3344 → 3345, and 3351 → 3353 cm−1. Peaks at 1592 and 3699 cm−1 disappeared, while new peaks emerged at 991, 3366, and 3382 cm−1. The peaks at 808 and 895 cm−1 correspond to the Iα and Iβ cellulose allomorphs [66] and the β-glycosidic linkage between glucose units, which form the backbone of cellulose and hemicellulose [67], respectively. The bands at 1155, 1367, and 1418 cm−1 are associated with xylan, representing anti-symmetric C–O–C stretching, phenolic OH bending, and CH2 symmetric bending in the xylan chain [68,69,70,71,72]. Mannan contributions are observed at 1020, 2892–2919, and 3344–3362 cm−1, corresponding to C–O stretching, symmetric CH2 stretching, and O–H vibrations arising from intra-chain (C2–OH···O5, C6–O···OH) and inter-chain (C3–OH···O–C6) hydrogen bonding, respectively [66,73,74,75]. The bands at 1227, 1265, 1330, 1454, 1508, and 1592 cm−1 indicate lignin, corresponding to C–OH stretching of phenolic OH groups, C–O stretching, C–O bending of aromatic rings, aromatic ring vibrations, C=C aromatic skeleton vibrations, and water absorption, respectively [68,73,76,77,78,79,80]. The peak at 1642 cm−1 is attributed to bound water [81], while the hydrogen-bonded OH stretching appears at 3752 cm−1 [82]. Overall, the lignocellulosic residue in the ALE film resembles lignocellulose derived from soyhulls [43,50] and cellulose obtained from corncob [48], soyhull [49], cow dung [55], grapevine [56], and alfalfa [44], with the addition of residual lignin components.

3.2.2. Antioxidant Property

The radical scavenging activity (RSA) and 50% inhibitory concentration (IC50) of the ALE film were found to be 5.88 ± 0.51% and 0.38 ± 0.04 g/mL, respectively. The IC50 value is slightly lower than that reported for alfalfa cellulose films [44], probably due to lignin retention in the lignocellulosic extract. The film’s ability to neutralize free radicals arises from the antioxidant activity of its inherent bioactive compounds [83,84]. The polyphenolic polymer with aromatic units in the lignin fraction significantly contributes to the antioxidant activity of the film [85]. In conventional active packaging systems, antioxidant compounds are usually added to improve food preservation. However, the natural antioxidant activity of the ALE film enables the development of active packaging without additional additives, providing a cost-effective solution. This property bears great potential to extend the shelf life of fresh produce by reducing oxidative degradation.

3.2.3. Hydration Properties

The ALE film exhibited a moisture content of 10.9 ± 0.3% and a water solubility of 50.9 ± 1.2%, values comparable to other cellulose-based films such as corncob (10.1% and 64.1%) [48], soyhull (8.4% and 69.6%) [49], switchgrass (11.3–17.8% and 48.3–71.2%) [59,60], cow dung (9.9–12.3% and 51.7–67.9%) [55], banana peel (13.7–22.3% and 24.5–65.7%) [52], and wheat straw (14.4–24.3% and 28.7–56.9%) [61].
In addition to moisture content and water solubility, hydrophobicity is a critical parameter for packaging applications. The ALE film exhibited a water contact angle of 78.9 ± 2.3° at 0 s, gradually decreasing to 69.9 ± 0.5°, 60.9 ± 1.5°, and 54.5 ± 0.4° at 10, 20, and 30 s, respectively (Figure 3a). These values are slightly higher than those reported for alfalfa cellulose [44] and soyhull lignocellulose films [43,50], and comparable to xyloglucan incorporated cellulose nanosphere film [86]. The presence of lignin hinders water penetration and increases the hydrophobicity [87,88]. The gradual decrease in contact angle is attributed to the inherent absorption, spreading, and swelling behavior of biopolymers [89,90,91,92]. The moderate hydrophilicity of the film makes it suitable for packaging moisture-insensitive products such as fresh fruits and vegetables.
The water absorption of the film was 45.3 ± 1.5% at 5 min, increasing to 51.8 ± 0.3%, 55.6 ± 0.2%, 59.9 ± 0.7%, 63.9 ± 1.5%, 67.6 ± 1.8%, and 69.3 ± 1.0% at 10, 15, 30, 60, 90, and 120 min, respectively (Figure 3b). The water absorption was lower than that of alfalfa cellulose [44], likely due to the hydrophobic nature of lignin, which fills voids within the fiber structure and limits water penetration [87,88]. Water absorption kinetics were analyzed using nine established models (Table 4). Among these, the Czel and Czigany, Singh, Pilosof, Gornicki, and Peppa models showed high R2 values (0.8982–0.9898), indicating a good fit. However, the Peleg model provided the best fit, with an R2 of 0.9990 and RMSE of 0.0179. In this model, K1 and K2 represent the initial water absorption rate and the maximum water absorption capacity, respectively. Similar trends for the Peleg model have been reported for cellulose-based films derived from corncob [48], soyhulls [43,49,50], alfalfa [44], grapevine [56], cow dung [55], and spent coffee grounds [58].

3.2.4. Soil Biodegradation

The soil biodegradation of the ALE film was evaluated under conditions of 24% soil moisture. The film showed a relatively slow degradation during the initial phase, reaching 44.9 ± 0.7% by day 19 (Figure 3c). During this period, the degradation progressed as follows: 5.0 ± 0.4%, 7.6 ± 0.1%, 11.8 ± 0.7%, 15.6 ± 1.0%, 18.9 ± 1.1%, 25.8 ± 0.5%, 30.9 ± 1.5%, and 39.4 ± 0.7% on days 3, 5, 7, 9, 11, 13, 15, and 17, respectively. This slower initial disintegration likely reflects the time required for microbial colonization and enzymatic activity. Further research is necessary to confirm this idea. Afterward, the degradation accelerated, reaching 90.1 ± 1.6% within the following 10 days, with values of 55.4 ± 1.5%, 70.2 ± 0.9%, 76.5 ± 1.0%, and 83.5 ± 1.8% on days 21, 23, 25, and 27, respectively. The biodegradation timeline of ALE film is comparable to that reported for avocado peel fiber [51], banana peel fiber [52], corncob cellulose [48], oat straw fiber [93], and wheat straw fiber films [61].
The biodegradation kinetics were analyzed using first- and second-order reduction reaction models, with the second-order model providing the best fit (R2 = 0.9969, RMSE = 0.0493) (Table 4). Using this model, the film’s half-life was calculated to be 19.4 days. Similar second-order kinetics and half-lives have been reported for lignocellulosic films made from soyhull [43,50], switchgrass [59,60], and spent coffee grounds [58]. The demonstrated soil biodegradability of the ALE film highlights its potential as an eco-friendly packaging option, unlike traditional plastics, which require specialized composting facilities for disposal.

3.3. Recovery of Chemicals

To make the process more environmentally friendly, the method of recovering used chemicals is ongoing. The 500 mL of ethanol used for each film production is recovered using a rotary vacuum evaporator, with approximately 92% successfully reclaimed. The remaining 8% consists of a mixture of ethanol and washed-off ZnCl2 and CaCl2 from the film. Similarly, NaOH is rinsed away with water during the lignocellulosic extraction. Current experiments focus on developing methods to recover these chemicals, aiming to make the film-making process entirely green process.

4. Conclusions

The current study demonstrates the green valorization of alfalfa biomass as a sustainable approach for producing biodegradable lignocellulosic films for packaging applications. Through systematic optimization of the proportions of alfalfa lignocellulosic extract (ALE), CaCl2, and sorbitol, sturdy films with a balanced combination of mechanical, barrier, and functional properties were developed. The optimized films were strong, fairly flexible, and transparent, and exhibited notable UV-light-blocking capacity and antioxidant activity. Additionally, the films showed favorable hydration behavior, making them suitable for moisture-insensitive food packaging. They biodegraded quickly in soil, with over 90% disintegration within 29 days at 24% soil moisture. These combined features position ALE films as a promising alternative to traditional petrochemical plastics, with the potential to lessen environmental burdens associated with packaging waste. Overall, this work provides a comprehensive framework for upcycling agricultural residues, such as alfalfa, into valuable biomaterials. The findings promote sustainable packaging development and support the circular bioeconomy, aiding environmental protection, income diversification for farmers, and innovation in sustainable packaging materials. Our future studies will explore scaling strategies, coating and blending approaches, and performance testing of films under real storage conditions to further improve industrial use.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/app152211889/s1, File S1: Equations.

Author Contributions

Conceptualization, S.J.; methodology, S.P. and S.J.; software, S.P.; validation, S.P.; formal analysis, S.P.; investigation, S.P.; resources, S.J.; data curation, S.P.; writing—original draft preparation, S.P.; writing—review and editing, S.J.; visualization, S.P.; supervision, S.J.; project administration, S.J.; funding acquisition, S.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Alfalfa & Forage Alliance, USDA Agriculture Research Service, agreement number 58-5010-3-012, and USDA National Institute of Food and Agriculture, SD00G677-20, 2021-67022-33469, and SD00H772-22.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

Acknowledgments

We thank Phil Taecker for the generous supply of alfalfa from his farm, Ann Taecker for help with biomass procuring and processing, and Kasiviswanathan Muthukumarappan for access to the Texture Analyzer and Todd Letcher for access to FTIR.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Applsci 15 11889 g001
Figure 1. The effects of independent variables alfalfa lignocellulosic extract (ALE) and CaCl2, ALE and sorbitol, and CaCl2 and sorbitol on Tensile strength (TS) (a–c), Elongation at break (EB) (d–f), and Water vapor permeability (WVP) (g–i), are displayed in the 3D contour plot diagram. The red points in the plot represent the points used in the Box-Behnken Design.
Figure 1. The effects of independent variables alfalfa lignocellulosic extract (ALE) and CaCl2, ALE and sorbitol, and CaCl2 and sorbitol on Tensile strength (TS) (a–c), Elongation at break (EB) (d–f), and Water vapor permeability (WVP) (g–i), are displayed in the 3D contour plot diagram. The red points in the plot represent the points used in the Box-Behnken Design.
Applsci 15 11889 g001
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Figure 2. (a) The alfalfa lignocellulosic extract (ALE) film’s light transmittance (blue line) and absorbance coefficient (red line), (b) FTIR spectrum of ALE with major peaks, and (c) FTIR spectrum of ALE film.
Figure 2. (a) The alfalfa lignocellulosic extract (ALE) film’s light transmittance (blue line) and absorbance coefficient (red line), (b) FTIR spectrum of ALE with major peaks, and (c) FTIR spectrum of ALE film.
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Figure 3. ALE film’s (a) water contact angle, (b) water absorption, and (c) biodegradation, with the function of time.
Figure 3. ALE film’s (a) water contact angle, (b) water absorption, and (c) biodegradation, with the function of time.
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Table 1. The film combinations (Coded: A, B, and C represent ALE, CaCl2, and sorbitol with values −1, 0, and 1 representing lower, mid, and higher values in their range; Actual: Alfalfa Lignocellulosic extract (ALE, g), CaCl2 (mM), and Sorbitol (%)), with their response Tensile strength (TS, MPa), Elongation at break (EB, %), and Water vapor permeability (WVP, 10−10 g m−1 s−1 Pa−1).
Table 1. The film combinations (Coded: A, B, and C represent ALE, CaCl2, and sorbitol with values −1, 0, and 1 representing lower, mid, and higher values in their range; Actual: Alfalfa Lignocellulosic extract (ALE, g), CaCl2 (mM), and Sorbitol (%)), with their response Tensile strength (TS, MPa), Elongation at break (EB, %), and Water vapor permeability (WVP, 10−10 g m−1 s−1 Pa−1).
RunIndependent VariableResponse
CodedActual
ABCALECaCl2SorbitolTSEBWVP
ALE1−1−100.320012.4 ± 0.46.7 ± 0.91.6 ± 0.1
ALE21−100.520014.9 ± 0.85.6 ± 0.51.3 ± 0.3
ALE3−1100.350015.4 ± 0.15.5 ± 1.31.2 ± 0.1
ALE41100.5500112.6 ± 0.32.9 ± 0.70.9 ± 0.1
ALE5−10−10.33500.52.2 ± 0.79.1 ± 0.11.3 ± 0.1
ALE610−10.53500.510.0 ± 2.64.2 ± 1.01.1 ± 0.2
ALE7−1010.33501.52.6 ± 0.46.3 ± 1.41.3 ± 0.1
ALE81010.53501.510.1 ± 1.06.6 ± 1.81.2 ± 0.1
ALE90−1−10.42000.54.8 ± 1.68.1 ± 1.21.3 ± 0.3
ALE1001−10.45000.59.6 ± 0.85.8 ± 0.11.0 ± 0.1
ALE110−110.42001.54.5 ± 0.56.0 ± 0.61.4 ± 0.1
ALE120110.45001.58.9 ± 1.64.8 ± 1.01.1 ± 0.1
ALE130000.435018.6 ± 1.15.3 ± 1.71.1 ± 0.2
ALE140000.435017.7 ± 0.15.9 ± 1.81.2 ± 0.2
ALE150000.435018.3 ± 1.56.4 ± 1.91.2 ± 0.1
Table 4. Water absorption and biodegradation kinetics model fitting of ALE film. (ND denotes “Not Detected”).
Table 4. Water absorption and biodegradation kinetics model fitting of ALE film. (ND denotes “Not Detected”).
ModelabcR2RMSE
Water absorption
Peleg (a as K1 and b as K2)0.06060.0141-0.99900.0179
Gornicki0.36710.35510.19380.98980.0082
Pilosof0.36710.355114.53540.98980.0082
Czel and Czigany (b as m)0.38150.1277-0.98290.0107
Peppas (a as K1 and b as K2)−0.02160.3045-0.90730.1495
Singh−3.489213.60143.27280.89820.0258
Garcia Pascual0.59191.2782-0.34140.2668
Vega-Galvez1.24080.7426-00.0808
Weibull (a as α and b as β)53,687,092.211.2069-NDND
Biodegradation
First-order (a as m)0.1084-1.65570.98080.1740
Second-order1.10310.2011−0.00290.99690.0493
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Paudel, S.; Janaswamy, S. Green Valorization of Alfalfa into Sustainable Lignocellulosic Films for Packaging Applications. Appl. Sci. 2025, 15, 11889. https://doi.org/10.3390/app152211889

AMA Style

Paudel S, Janaswamy S. Green Valorization of Alfalfa into Sustainable Lignocellulosic Films for Packaging Applications. Applied Sciences. 2025; 15(22):11889. https://doi.org/10.3390/app152211889

Chicago/Turabian Style

Paudel, Sandeep, and Srinivas Janaswamy. 2025. "Green Valorization of Alfalfa into Sustainable Lignocellulosic Films for Packaging Applications" Applied Sciences 15, no. 22: 11889. https://doi.org/10.3390/app152211889

APA Style

Paudel, S., & Janaswamy, S. (2025). Green Valorization of Alfalfa into Sustainable Lignocellulosic Films for Packaging Applications. Applied Sciences, 15(22), 11889. https://doi.org/10.3390/app152211889

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