Lignin derived from Borassus flabellifer leaves as a nano-filler for polyhydroxybutyrate composites

Abstract

Deterioration of the environment is occurring at an alarming rate due to the widespread use of plastics. Conventional plastics do not degrade easily and pose severe environmental threats. Hence, it is imperative to devise a sustainable solution to reduce their detrimental impact on our ecosystem. The applications of polyhydroxybutyrate (PHB) are limited due to its shortcomings, such as low tensile strength, poor thermal stability, and inherent brittleness. In the present study, lignin, another natural polymer, was blended with the polymer PHB to address these limitations. Lignin, extracted from the leaves of Borassus flabellifer, is a complex polymer second only to cellulose in abundance. It possesses excellent mechanical properties, good toughening capacity, and disease resistance in plants, making it a promising co-polymer for PHB. Upon blending the polymers PHB and lignin, the composite film was further assessed for structural, mechanical, and thermal analysis. Incorporation of 20% lignin into PHB increased the tensile strength from 0.5 MPa (pure PHB) to 1.05 MPa. Water absorption after 24 h was reduced from 13% in PHB to 10% in the composite, indicating improved hydrophobicity due to effective lignin integration. Enhanced thermal properties were also observed, with the composite film showing a degradation temperature of 300°C, compared to 290°C for PHB alone. These findings affirm the improved thermal stability and durability of the composite film. Furthermore, PHB-lignin composite films hold strong potential for applications in sustainable food packaging systems as the polymers involved are entirely bio-derived and bio-stable. The approach demonstrates a simple yet effective method for enhancing biopolymer performance using plant-derived additives. The developed composite offers a promising alternative to synthetic packaging materials, aligning with global sustainability goals.

Graphical Abstract

Keywords

Biopolymers polyhydroxybutyrate lignin PHB-lignin composites

Introduction

Natural fiber is vital in the development of green composites, which aid in the resolution of ecological and environmental issues.¹ Lignin, as an inherent aromatic polymer within plant cell wall structures, holds a fundamental function in shaping plant growth and exerts a pronounced influence on the effective utilization of sustainable lignocellulose biomass.² Biopolymers are polymers found naturally in living organisms. A biopolymer is a long-chain molecule composed of monomeric components covalently linked together to form a biodegradable molecule. The principal sources of biopolymers are plants, trees, microorganisms, and other natural resources. Biopolymers are highly organized molecules with clearly defined three-dimensional structures, whereas synthesized polymers are less complex and more arbitrary.³ Polyhydroxyalkanoate (PHA) is used as a prime illustration of a natural, biodegradable polymer, produced by various strains that can be both Gram-negative and Gram-positive. In the past few years, PHAs have attracted considerable interest from experts worldwide due to their multiple beneficial characteristics. Moreover, it is biocompatible, biologically degraded, tested, and used as a substitute for the normal day-to-day use of conventional plastics.⁴ PHB belongs to the PHA class and is the primary focus of this study. The microbial biosynthetic pathway of PHB is understood when two molecules of acetyl-CoA undergo a condensation process, giving aceto-acetyl-CoA. On reducing the acetoacetyl CoA, it attains hydroxy butyryl CoA, a monomer. This hydroxy butyryl CoA is later polymerized to obtain PHB.⁵ Some of the exciting properties of PHB are that they are insoluble in water, resist degradation by hydrolysis, are non-toxic, have excellent permeability for oxygen, and are biostable and biocompatible. Even though it has many desirable properties, it also comes with a handful of disadvantages. To name a few, it is highly brittle, has negligible tensile strength, inconsiderable thermal stability, and exhibits high fragility.⁶

The co-polymer used for this research is lignin, and its properties were considered, given that incorporating a co-polymer with the compound PHB made the polymer stronger and more durable. It is also fascinating to accept that lignin is the second most abundant organic polymer found on the planet, following cellulose. Lignin’s structure is highly branched, and it follows a phenylpropanoid pathway.⁷ One of the most interesting characteristics of lignin, which makes it an ideal co-polymer to be blended with PHB, is that it showed excellent resistance to degradation as it was bound by strong chemical bonds. The key reason for this is due to the substantial amount and high adhesion of hydrogen bonds.⁸ Most of the time it acts as a filler material in the cell walls of plants and accounts for remarkable and outstanding mechanical strength.⁹ Hence, they render the plants the necessary support and make them stand upright. The amino acid phenylalanine acts as a precursor; it plays a major role in the transportation of fluids and water conduction mechanisms in plant stems. They possess excellent mechanical properties and act as nano-fillers.^10,11

The lignin was extracted from well-dried leaves of Borassus flabellifer, conventionally known as palm leaves. The selection of the source had been validated by the fact that the leaves were less explored and the lignin from its leaves has ample desirable properties. B. flabellifer comes under the Aceraceae family and is mainly found in regions like Sri Lanka, Bangladesh, Africa, and India.¹² The leaves and the stalk of B. flabellifer have been used for various domestic purposes, including fuel for cooking, fence and fan making, and writing materials.¹³ This study focuses on the extraction of lignin from B. flabellifer leaves, blended with PHB made into thin films acting as a nanofiller for PHB-composites, and further explores its physical, mechanical, and thermal properties.

PHB-lignin composites are a remarkable candidate material for food packaging applications based on their desirable mechanical properties, biodegradability, and moisture uptake properties. Lignin is one of the most abundant biopolymers on the planet with an aromatically rich composition, and increasing lignin content in the PHB matrix deals with increasing reactivity as it plays an important role in enhancing thermal stability and barrier properties of PHB-lignin composites, which are good to prolong the freshness of food and shelf life.¹⁴ Lignin has a significant degree of intermolecular hydrogen bonding and a rigid aromatic structure, therefore decreasing water permeability of the films and increasing mechanical performance. Moreover, lignin has natural antioxidants and antimicrobial properties that could provide additional active packaging advantages, such as greater protection from microbial growth and oxidative spoilage. From an economic perspective, lignin is a low-cost and sustainable secondary product from the agro-industrial sciences, which provides PHB-lignin composites with an additional value as a biopolymer.¹⁵ The integration of lignin with PHB is intended to improve physicochemical, antimicrobial, and mechanical properties that provide opportunities for the development of next-generation biodegradable packaging systems for food packaging.^14,15

Experimental

Materials

A commercial poly (3-hydroxybutyrate) (PHB) powder (average Mn 50,000 Da, Source MKCR0793) was supplied by Sigma Aldrich, Chloroform extra pure, 99% (stabilized with 1-2% v/v ethanol), Leishman Stain Solution L0060 were purchased from Southern India Scientific Corporation. B. flabellifer leaves collected from a neighboring local street to extract the lignin.

Extraction of lignin

Decontamination and extraction of lignin from B. flabellifer leaves were done in a systematic manner using solvent-assisted extraction to obtain pure leaves with adequate lignin recovery.¹ The harvested leaves were swept to remove dirt and soil-soluble surface impurities. The leaves were spread in front of direct sunlight and allowed to dry naturally during the darkest part of the day for 24-96 h / day at the sites studied. This air-drying took place for 1 to 2 days to produce concentrated moisture content in leaves and to initiate the breakdown of soft tissues. After sun drying, the leaves were immediately exposed to a thermal treatment for 3 h in a hot air oven at 80°C. Heat release was required to further reduce moisture content and neutralize microbial contaminants. The treated leaves were allowed to dry at a room temperature of about 36°C for 15 days to allow solvents or volatile impurities to leach off. Lignin was extracted using the alkali-based delignification process.¹⁶ For this treatment, dried and conditioned leaf biomass was crushed into smaller particles to increase the surface area of the solid, and a solution of 4% (w/v) sodium hydroxide (NaOH) was prepared with the help of NaOH pellets. The residue was mixed with NaOH solution in a 1:10 ratio, where 1g of residue to 1 mL of NaOH solution. The solution was heated in a water bath at 120°C for 2 h. During this time, the inhospitable alkaline conditions allowed dissolution of ester and ether bonds in the lignocellulosic matrix, resulting in the release of lignin into the liquid. The dark brown to black liquor that was derived from the delignification was filtered through Whatman filter paper No. 1.^17,18 The filtrate was dried at room temperature for 48 h to obtain powdered lignin. Spectral analysis of the final lignin product was performed using ¹H NMR and FT-IR characterization.^19,20

Composite film preparation

The solvent-assisted method was used to prepare the biodegradable PHB-lignin composite film with better interfacial compatibility and dispersibility. In this method, the PHB and lignin were weighed out in an 80:20 wt ratio optimized based on water contact angle (WCA) measurements to compromise between hydrophobicity, integrity, and surface wetting. PHB was dissolved in chloroform, a suitable, volatile, non-polar solvent for hydrophobic polymers such as PHB, while lignin was dissolved in methanol because of its polar and phenolic components.²¹ PHB-chloroform and lignin-methanol solutions were made separately in Falcon tubes and sonicated with an ultrasonic probe. Sonication was performed for 15 mins to ensure the phytochemicals were uniformly dispersed and reduce aggregate particle sizes of lignin for a more homogenized composite blend. The two solutions were vortexed at low speed for 15 minutes for blending and contact at the molecular at nanometer scale level between PHB and lignin. After a thorough mixing, the mixed solution was cast into a clean petri dish for drying in the fume hood at room temperature to obtain the dried PHB-lignin composite film.²²

Characterizations

Nuclear magnetic resonance (NMR) analysis

By examining the magnetic field’s interaction with a specific sample, NMR spectroscopy can determine the characteristics of organic molecules and provide details about their fundamental structure and molecular conformation. The extracted lignin was liquified in 600 μL of chloroform and then analyzed by the ¹H NMR spectroscopy (Bruker).¹⁸

Fourier-transform infrared (FT-IR) spectroscopy analysis

The synthesized PHB-lignin composites, standard PHB, and lignin were analyzed by Agilent Cary 660 FT-IR spectroscopy in the range of 400 and 4000 cm⁻¹ to compare their IR spectra.¹⁸

Scanning electron microscope (SEM) analysis

The morphology of the extracted PHB-lignin composite was visualized with the help of Quanta200 FEG FE-SEM, a field-emission scanning electron microscope. The ‘Bruker 129 eV′ detector was also used to conduct energy dispersive X-ray spectroscopy (EDS) investigation.²⁰

X-ray Diffraction (XRD) analysis

XRD analysis was performed for PHB, lignin, and the PHB-lignin composites. It was performed at 37 °C using a generator and a Cu-K source with a wavelength of 1.54,060 and 15 mA, respectively.²¹

Atomic force microscopy (AFM) analysis

PHB and the extracted PHB-lignin composite were given for the analysis of AFM to compare their surface morphology and surface roughness.²²

Thermal and mechanical property analysis

Samples were taken and heated at a rate of 20°C per minute and scanned at a temperature difference of 30°C to 800°C with the help of a Thermogravimetric analyzer (TGA) to check the thermal degradability. Samples were taken and subjected to heat at a rate for performing DSC analysis. Mechanical properties such as the tensile strength and Young’s modulus of neat PHB and PHB-lignin composites were further determined.^23,24 The activation energy was calculated by the equation (1)

k = A e \frac{- E a}{R T}

(1)

Where, k = rate constant,

T = absolute temperature,

A = frequency factor,

E_a = molar activation energy,

R = universal gas constant.

Water contact angle (WCA)

The water contact angle was calculated by the sessile drop technique. A drop of water was placed on the film, and the angle was measured from it. The angle found was the water contact angle which measures the wettability of the film.²⁵

Water absorption test

A water absorption test was carried out on PHB and PHB-lignin composite films with measurements of 2 × 3 cm. The films were weighed before soaking in distilled water at room temperature for 24 hours. Water absorption was measured by the equation (2).²⁵

Water absorption (%) = \frac{Final weight - Initial weight}{Initial weight} \times 100

(2)

Biodegradability analysis

By using the soil burial method, the biodegradability of the pure PHB and PHB-lignin composite films was evaluated. The samples were weighed first and then buried in the soil. Each film was inserted into the soil and extracted in 10 days for a month. Then the retrieved sample was cleaned with water and dried in an oven. Biodegradability was assessed by determining the weight loss of the samples using the following equation (3).²⁵

Water loss (%) = \frac{Initial weight - Final weight}{Initial weight} \times 100

(3)

Results and discussion

NMR analysis

The chemical structure and atomic arrangement of the lignin were elucidated using 1H NMR or proton NMR analysis.²¹ The spectral range was maintained at 0-11 ppm. Conspicuous peaks or spectra at 0.808, 1.21, and 2.50 ppm which were obtained showed the terminal methyl protons of lignin side chains, methylene protons, and methyl groups, respectively; these chemical shifts correspond to earlier established ranges for lignin. Given the singlet peaks from methyl and methylene protons and their observations as singlets indicate a relatively uniform environment from each separate dipolar interaction.^14,26 The spectral profile obtained matches traditional ¹H NMR for lignin and was representative of a given aliphatic and aromatic proton environment in the lignin structure. Such characteristic peaks are important for understanding the structural and reactivity of lignin. Dimethyl sulfoxide (DMSO) functions in a way that preferentially dissolves the lignin and works with proton NMR as a solvent substitution, due to limited overlap in proton NMR.¹⁷ Every peak obtained was a singlet. A sharp peak at 3.53 indicated DMSO, which was used as a solvent for analysis (Figure 1). The peaks obtained were observed to be similar to standard NMR analysis of lignin.²⁷

Figure 1.

¹H NMR spectroscopy analysis of lignin particles extracted from B. flabellifer leaves

FT-IR analysis

The band at 3367.71 cm⁻¹ referred to the O-H functional group and showed the presence of the hydroxyl (alcohol) group. The peak at 2929.87 cm⁻¹ indicated aliphatic C-H vibrations, signifying the presence of methyl and methylene groups. 1624.06 cm⁻¹ peak referred to the carbonyl (C = O) group, indicating the presence of carbonyl groups in the structure.²⁸ The absorption band at 1514.12 cm⁻¹ was attributed to the C = C stretching of the aromatic rings in lignin. The band at 1454.33 cm⁻¹ corresponded to aliphatic C-H stretching vibrations, providing additional evidence for the presence of aliphatic groups. Finally, the absorption bands of 964.41 cm⁻¹ and 785.03 cm⁻¹ were related to C-O stretching vibrations, namely aliphatic ether bonds seen in lignin. The presence of hydroxyl groups, aliphatic and aromatic moieties, carbonyl groups, and aliphatic ether linkages was shown by FTIR analysis of the lignin sample, confirming the complex composition of the lignin (Figure 2(a)). The polymer’s functional groups have been identified by the wide band at 3437.15 cm⁻¹ indicated -OH stretching groups, whereas 2980.02 cm⁻¹ and 2931.80 cm⁻¹ represented C-H stretching groups, respectively. The 1750 cm⁻¹ absorption band corresponded to the C = O ester bond stretching. The band at 1452.40 cm⁻¹ related to C-H of the methylene group. The next peak is 1375.25 cm⁻¹, which confirms the presence of the alkane group. Similarly, in the peak 1307.16 cm-1, C-O stretching, and in peak 1095.57 cm-1, the C-O stretching of the aliphatic group was visible. The band observed between 1740 cm⁻¹ and 1748 cm⁻¹, has been identified as carbonyl (C = O) ester bond vibrations (Figure 2(b)). The bands detected match those reported by other studies.²⁹ Comparing the images in (Figure 2(c)), the first image confirmed the sample as lignin, and the second image confirmed that the peaks shown were indicating the PHB. Combining both images, the peaks at 2972.31 cm⁻¹ and 2927.94 cm⁻¹ were assigned to the stretching vibrations of aliphatic C-H bonds, indicating the presence of alkyl groups in both the PHB and lignin components of the composite. A peak observed at 1750 cm⁻¹ corresponds to the stretching vibrations of carbonyl (C = O) bonds, suggesting the presence of carbonyl groups, such as esters or ketones, within the PHB component of the composite. The peak at 1452.40 cm⁻¹ referred to the bending vibrations of CH₂ bonds and provided evidence for the presence of methylene groups in both PHB and lignin. The peak at 1514.12 cm⁻¹ had been associated with the stretching vibrations of C = C bonds, indicating the presence of aromatic rings within the lignin component of the composite. Lastly, the peak at 457.13 cm⁻¹ represented the bending vibrations of C-H bonds in the aromatic rings of lignin.¹⁷

Figure 2.

FT-IR analysis of (a) extracted lignin particle from B. flabellifer leaves, (b) PHB film, and (c) PHB-lignin composite film

SEM analysis

The PHB and the PHB-lignin composite films were analyzed by SEM. The analysis showed a thin film structure with numerous pores in the film for the PHB film (Figure 3(a)). The pores and shape of the PHB compound were evident at 10,000x magnification.³⁰ reached a similar conclusion on this point. (Figure 3(b)) illustrated a smooth visible surface, blocking the pores of the PHB in the composite film. The energy-dispersed X-ray results are shown in Table 1 Which revealed that the carbon content in the PHB film was 4.33% more than the composite film, while the oxygen content in the PHB film was 4.01% less than the composite film. Comparing both the SEM images, it is evident in (Figure 3(b)) that there were no pores and with the help of the EDX table, it was concluded that the lignin had been successfully incorporated in the PHB film as a composite, which was found to be comparable to.⁸

Figure 3.

SEM analysis of (a) PHB film and (b) PHB-lignin composite film

Table 1.

EDX analysis of (a) PHB film, (b) PHB-lignin composite film

XRD analysis

The X-ray Diffraction pattern (Figure 4(a)) displayed distinctive peaks indicating a crystalline structure. These peaks represented the crystalline nature of lignin. The peak at 2θ = 27° is related to a distinct arrangement of atoms or molecules in lignin’s crystalline regions. The 2θ = 45° peak at 1500 intensity showed the presence of different crystallographic arrangements inside the lignin structure. The position and strength of this peak helped to characterize the crystalline structure of the lignin. The intensities of these peaks revealed the crystallographic structure of lignin molecules.³¹ The peaks were found to be very intense at 2θ = 13.7° and 2θ = 17° showing the PHB is semi-crystalline in nature. The peak at 2θ = 17° further confirmed the existence of crystalline regions in PHB and the arrangement of PHB molecules. In (Figure 4(c)), the XRD spectrum lacked strong peaks or identifiable diffraction patterns, indicating that the composite lacked a well-defined crystalline structure. Instead, the scattering pattern was observed wide, indicating that the molecular components were disorderedly arranged. Since the PHB-lignin composite is amorphous, the polymer chains of both PHB and lignin were randomly oriented and lacked long-range order. This is due to variables such as the blending process or the interaction between PHB and lignin, which interfered with the production of crystalline domains (Figure 4(b)). A similar outcome was obtained.³²

Figure 4.

XRD analysis of (a) lignin particles extracted from B. flabellifer leaves, (b) PHB film, and (c) PHB-lignin composite film

AFM analysis

AFM analysis was employed to visualize the topography of the surface (or surface morphology) at the atomic level. (Figure 5(a)) shows the AFM image of PHB film, the emptiness, and the scattered distribution of pores were visible. The PHB film has an average roughness of the surface (Ra) of approximately 1.12 µm, signifying a rough texture, with a variety of hollows and peaks. Whereas, in the AFM image of PHB-lignin composite film (Figure 5(b)), the number of pore spaces was reduced, and the pore spaces were penetrated by lignin particles.³³ The addition of lignin significantly reduced both pore numbers and sizes, resulting in a denser material with more uniform morphological features. The Ra was reduced to 0.59 µm, indicating improved surface uniformity and reduced topographical variability.³³ The improvement is attributed to the homogenous distribution of lignin fragments in the PHB matrix, which improved interfacial interactions and added strength to the composite film. The morphological refinement seen here is a result of the successful inclusion of lignin as both an additive compatibilizer and a surface modification, which finally results in improved surface characteristics and composite durability.³⁴ Hence, the integration of the co-polymer was morphologically confirmed.³⁵

Figure 5.

AFM analysis of (a) PHB film and (b) PHB-lignin composite film

Thermogravimetric analysis

The thermogravimetric analysis was performed to study the thermal stability of the films. The thermogram (Figure 6) showed the thermal degradation of PHB and PHB-lignin composite film. In the PHB film, the first weight loss of 95% was visible at 275 °C with a loss of water weight of 5%. After this, an immediate loss of weight of 3% was visible at 490 °C. This was due to the degradation of the thermolabile components. The highest amount of decomposition of 15.5% weight was observed at 310 °C, thus concluding that the degradation temperature was 300 °C. The first weight loss of 90% for the PHB-lignin composite film was identified at 275 °C. The temperature of degradation for the composite film was recorded at 315 °C with a weight loss of 13%.³⁶ reported that the decomposition temperature for PHB was 235 °C, and for the PHB-lignin nanofibers, it was 240 °C. In a previous study,⁸ found that the PHB was entirely degraded at 260°C, while the reduction of weight was 60% at a similar temperature. Thus, in conclusion, lignin enhanced the thermal stability of the PHB film. To further characterize thermal stability, activation energy (Ea) was calculated using the Arrhenius equation on TGA curves at various heating rates. The activation energy for neat PHB was calculated to be 115.7 kJ/mol. Whereas, the Ea for the PHB-lignin composite was determined to be 120.5 kJ/mol, which indicates better thermal durability of the PHB-lignin composite.³⁶ Thus, the combination of the reinforcing and thermal stabilising role of lignin and its increase in degradation profile and thermal performance of PHB films was significantly increased.³⁷

Figure 6.

TGA analysis of PHB and the PHB-lignin composite films

DSC analysis

The phase transitions, such as the glass-transition temperature, melting point, and crystallization temperature, were determined from the thermogram using DSC analysis. This analysis checks the polymer nature when heat is externally introduced during a phase transition. The melting temperature of the PHB film (Figure 7) was estimated at 290°C, whereas for the composite film, it was slightly higher, which was 300°C. At 256°C, which was the crystallization temperature, the melted PHB film developed a crystalline state upon cooling.³² The crystallization temperature for PHB-lignin composite film was recorded as 260°C, which was slightly higher than PHB. This proved that the lignin has improved the mechanical properties of PHB. Furthermore, at 180°C, the homogeneous PHB film was unheated to create a glass-like structure, which is known as the glass transition temperature. For the composite film, 185°C temperature was detected. The fact that both melting temperature and glass transition temperature were present indicated that the films contained both crystalline and amorphous parts. Applications like cohesiveness and packaging are avail of this characteristic.³² mentioned in the paper that the crystallization temperature for the PHB-lignin composite was 111°C, which is half of the temperature in this study.³⁸ demonstrated that lignin increased the number of spherulites produced in addition to acting like a nucleating agent and lowered the activation energy during the crystallization mechanism.

Figure 7.

DSC analysis of PHB film and PHB-lignin composite film

Tensile strength analysis

Studies of mechanical characteristics were conducted into a comparison between the PHB film and the PHB-lignin composite film. Different parameters, such as tensile strength (σ_y), Young’s modulus (E), and elongation at break (ε_b), were verified and formulated based on the stress-strain curve. From (Figure 8(a)), the tensile strength of the composite film gathered was 1.01 MPa, whereas the tensile strength of the PHB film attained 0.565 MPa. The composite film comparatively showed higher tensile strength. Likewise, the PHB film showed Young’s modulus value of 6.6 GPa, which was less than the Young’s modulus value for the composite film, 11.8 GPa. The enhancement of these mechanical properties in the composite film is solely attributed to the addition of a co-polymer lignin.³⁹ Therefore, lignin acts as a reinforcing nano-filler in PHB and strengthens the PHB matrix. (Figure 8(b)) The ε_b was slightly increased from the PHB film in comparison to the PHB-lignin composite film, from 8.50 % to 8.56% for both films, suggesting that the lignin did not embrittle the film. Indeed, in many reports, lignin may reduce the crystallinity of PHB, and this contributes to maintaining or even strengthening ductility, even when strength increases. The calculated ultimate force (N) was observed to be equally same for both films, that is, 1.27 N.¹¹ The above-represented parameters vividly indicate the strength, pliability, and elastic nature of the composite film made by lignin, and the drawbacks of PHB were masked.¹¹ The effect of lignin on mechanical properties is well known to depend on the dispersion and loading. With poor dispersion or an excess of lignin, biopolymers tend to become less ductile. The biopolymer composites composed of PLA-lignin material showed a significant loss in toughness; elongation of these composites was reduced by 82% compared with the control and loosened by 79%. Well-dispersed nanoparticles of lignin support the stiffness of a polymer.²³ This integration of lignin had a momentous impact on qualities like rigidity and strength of PHB.¹¹

Figure 8.

Mechanical testing of (a) PHB film and (b) PHB-lignin composite film

WCA measurement

The hydrophobicity of the film is an important criterion for its application in industries like food packaging. In general, contact angle readings below 65° suggested a hydrophilic nature, while readings above 65° imply a hydrophobic nature. From Figure 9, it was estimated that the water contact angle for the composite was found to be 78°.⁴⁰ reported similar results where softwood biorefinery lignin was integrated to make the composite film. The increased hydrophobicity can be attributed to the hydrophobic nature of lignin and the increased surface roughness from the incorporation of lignin. Other authors reported that lignin added to carboxymethyl cellulose films increased the WCA to 80.7°, improving the film for packaging purposes.⁴¹ The authors also reported that the addition of lignin nanoparticles to starch films had a high impact on increasing the WCA to more than 120°, greatly improving the water resistance. The hydrophobicity data suggested that lignin is a functional additive that lignin is a functional additive that can increase the hydrophobic properties of biopolymer films and support food packaging applications.⁴² The image and the readings showed that the composite film is hydrophobic and has a relatively small amount of wetting capacity, which made it well-suited for use in food packaging.⁴³

Figure 9.

WCA measurement of PHB-lignin composite film

Water absorption test

Water resistance in biopolymer films is important for packaging applications in products used in moisture-rich environments like food. In Table 2, the water absorption capacity of neat PHB was 13%, but the incorporation of lignin reduced the water absorption capacity to 10%. This was likely due to lignin’s hydrophobic nature, which limited the diffusion of water molecules into the polymer matrix. As an amorphous aromatic polymer, lignin has lower water absorption than some other natural fiber components, which produces a composite that has a higher resistance to moisture absorption.⁴² In addition, studies have shown that lignin in biopolymer matrices improved water resistance and mechanical properties, making the composite suitable for packaging applications. Therefore, it is evident that adding lignin to PHB films is a good approach to improving the water resistance of PHB films, which increases the potential industrial uses for PHB films, especially for products that require moisture barrier properties.^44,45

Table 2.

Water absorption test of PHB and PHB-lignin composite film

	Weight before absorbing water (mg)	Weight after absorbing water (mg)	% increase
PHB film	15	17	13
PHB-lignin composite film	6	6.6	10

Biodegradability analysis

The biodegradation rate of PHB and PHB-lignin composite films was assessed for 30 days to determine the extent of environmental degradability (Figure 10).^46,47 The biodegradation of the PHB film was slow and then increased, with weight loss % of 5.6% on day 10, 23.78% on day 20, and 65.78% by day 30. The PHB-lignin composite film experienced a greater rate of biodegradation with weight loss percentages of 9.09%, 54%, and 76%, respectively, for each of the same time points. Enhancing the biodegradation profile of the composite films can be attributed to adding lignin, which not only disrupts the crystalline structure of PHB, enhancing susceptibility of the polymer to microbial development and degradation. This has also been documented in other research studies, where biodegradable PHB matrices were demonstrated to have advanced biodegradation rates under compost conditions due to specific lignin content.⁴⁸ The biodegradability of PHB was significantly improved by adding lignin composites to the PHB matrix, indicating that the reinforcement is an added bio-composite benefit for environmentally sustainable applications. It was concluded that the composite film degraded much faster than the PHB film, which was reported in earlier studies.^39,48

Figure 10.

Biodegradability testing of PHB and PHB-lignin composite films over 30 days

Conclusion

The PHB-lignin composite, developed by combining bio-based components, aligns with global efforts to reduce plastic dependency and promote green materials. The improvement in hydrophobicity of the PHB-lignin composite, exhibited by the increased water contact angle, suggests that films can be used in novel carrying applications to wet-like environments found in food systems. Integration of lignin with PHB was significant and promising for potential applications in food packaging systems, as the lignin from this source acts as a UV-blocking agent and has oxygen permeability. Furthermore, complete biodegradability of the PHB-lignin composites under natural and industrial composting conditions makes them favorable from an environmental perspective, as increasingly more stakeholders in local and regional community systems, and society at large, include plastic pollution as pressing issues that require urgent solutions. Improved thermal degradation temperature suggests a more thermally stable composite, which may extend the shelf life and usability of the material in high-temperature conditions. The significant increase in tensile strength and thermal degradation temperature with lignin incorporation demonstrates its role in enhancing the structural integrity and heat resistance of PHB films, thus broadening their applicability in more demanding packaging environments. Overall, the results highlight the effectiveness of lignin as a reinforcing agent, enhancing the structural integrity and durability of the biopolymer film. However, the study was conducted at a laboratory scale, and further testing under industrial conditions is essential to validate long-term performance and stability. This approach not only adds value to agricultural waste but also contributes to the development of sustainable materials in line with circular economic principles. Moreover, the composite’s multifunctional properties make it a strong candidate for industrial-scale production, particularly in packaging and biomedical fields. Future investigations should focus on optimizing lignin concentration, assessing biodegradability over time, and evaluating barrier properties under different environmental conditions.

Footnotes

Acknowledgements

The authors sincerely thank the Department of Biotechnology, SRM Institute of Science and Technology, and Nanotechnology Research Centre, SRM Institute of Science and Technology, for providing research and analytical facilities.

ORCID iD

Radha Panjanathan

Informed consent

The authors have agreed to publish the paper.

Author contributions

A.B., S.G. and S.R. Formal analysis of the data and validation and preparation of the original draft, collection of resources such as protocols and other related research papers and review of the original draft; R.P. Conceptualisation, supervision and writeup revision – review and editing.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

The data used to support the findings of this study are available from the first author upon request.*

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