Biocompound and Lake Pigment Extraction from Invasive Alien Plant Biomass for Sustainable Ink Applications

Abstract

The management of invasive alien species (IAS) generates large amounts of plant waste biomass that is commonly disposed of by burning or destruction, leading to environmental and economic drawbacks. At the same time, the production of synthetic dyes and pigments used in printing and graphic applications remains a significant source of pollution. In this context, the valorization of IAS biomass as a source of natural colorants represents a sustainable alternative aligned with circular economy principles. Here, biocompounds and natural dyes were extracted from four invasive or non-native plant species—Arundo donax, Phytolacca americana, Tradescantia fluminensis, and Eucalyptus globulus—using five solid–liquid extraction methods: infusion, infusion with heat, thermal agitation, Soxhlet extraction, and ultrasonic-assisted extraction. Extraction efficiency and color preservation were comparatively evaluated. Although Soxhlet extraction provided the highest extraction yield (up to 30.5%), infusion with heat proved to be the most suitable method for preserving color integrity and minimizing oxidation. Liquid dyes obtained by the selected extraction method were converted into solid pigments through a lake pigment precipitation process using aluminum potassium sulfate and sodium bicarbonate. The resulting pigments were characterized in terms of chemical composition, particle size, and chromatic properties, and subsequently formulated into oil-based inks using linseed oil as binder. Scanning electron microscopy revealed pigment particle sizes ranging from approximately 2.1 to 8.3 µm, depending on the plant source, and confirmed adequate ink penetration and distribution on commercial printmaking paper. The obtained pigments exhibited color tones ranging from yellow to brown and grey, mainly associated with the phenolic and tannin content of the original biomass. Printing tests demonstrated the suitability of the developed inks for manual printmaking techniques, highlighting the potential of IAS-derived pigments as sustainable alternatives for artistic and printing applications.

1. Introduction

The proliferation of invasive alien species (IAS) represents a major environmental challenge, particularly in riparian and disturbed ecosystems, where their rapid expansion leads to biodiversity loss, soil degradation, and ecosystem imbalance [1,2,3,4]. Management strategies aimed at controlling IAS typically involve mechanical removal followed by disposal through burning or destruction, practices that generate environmental pollution and economic costs [5]. Consequently, the valorization of IAS-derived biomass has gained increasing attention as a sustainable approach to mitigate these impacts while promoting circular economy principles.

In parallel, the dyeing, pigment, and printing industries remain among the most polluting industrial sectors due to their reliance on synthetic colorants derived from petrochemical and heavy-metal-based compounds [6,7,8]. Although these materials provide high stability and color intensity, they contribute significantly to water contamination and pose long-term environmental and health risks. As a result, increasing interest has emerged in the development of bio-based and renewable alternatives for pigments and inks, particularly those derived from plant-based sources [9,10].

Plant-derived dyes and pigments originate from a wide range of biocompounds, including flavonoids, tannins, carotenoids, anthocyanins, chlorophylls, and other phenolic compounds, which are responsible for diverse chromatic properties in botanical sources [11,12]. These biocompounds fulfill multiple biological functions in plants while simultaneously determining their color expression, ranging from yellow and brown tones to red, purple, and blue hues.

Table 1. Major biocompounds present in botanical sources and their specifications on color characteristics.

Biocompounds	Specifications
Indigonoids	Present in the glucoside in the plant Indigofera tinctoria, responsible for a characteristic blue [13,14].
Tannins	Present in different parts, depending on the plant species (barks, stems, leaves and roots) and color varies from yellow, brown and reddish color shades [15].
Flavonoids	Present in most green plant species and responsible for flower coloring, for example from red to purple anthocyanins [14].
Quinonoids	Like the ones present in Henna [12].
Carotenoids	Present in plant and bacteria, allowing us to get yellow, oranges and red colors [11].
Betalains	Present in the Caryophyllales, cactus and some fungi and responsible for yellow and red coloring [11]. However, other studies [13] demonstrated that indigo (blue color) is also structurally related to betalains.
Chlorophyll	Present in all photosynthetic plants, responsible for green color. However, it is one of the most difficult colors to extract when dyeing [11].

summarizes the major classes of plant-derived biocompounds commonly associated with natural colorants and their corresponding chromatic characteristics, providing a chemical framework for the interpretation of the pigments obtained in this study.

Although natural dyes have historically been used in textile and artistic applications, their limited stability and solubility have restricted their broader adoption in printing and material-based technologies [16]. One strategy to overcome these limitations is the transformation of soluble dyes into insoluble pigments through lake pigment formation, a process based on the complexation of organic colorants with metal ions to improve durability and applicability in solid media [8,17,18].

Recent studies have demonstrated that several invasive and non-native plant species, including Arundo donax, Tradescantia fluminensis, Phytolacca americana, and Eucalyptus globulus, contain significant amounts of phenolic compounds and antioxidants, supporting their potential reuse in industrial and material applications [4,19]. However, despite this recognized chemical potential, the use of IAS-derived biomass as a source of solid pigments suitable for ink formulation and printing applications remains insufficiently explored. In particular, comparative assessments of solid–liquid extraction methods and their influence on color preservation, pigment formation, and ink performance are still scarce.

The present study addresses this gap by investigating the extraction of natural dyes from selected invasive and non-native plant waste biomass using five solid–liquid extraction methods: infusion, infusion with heat, thermal agitation, Soxhlet extraction, and ultrasonic-assisted extraction. The extracted dyes were subsequently converted into solid pigments via a lake pigment precipitation process and formulated into oil-based inks. The pigments and inks were characterized in terms of extraction efficiency, chromatic properties, chemical composition, particle size, and printing behavior on commercial paper substrates. By integrating invasive plant biomass into pigment and ink production, this work aims to contribute to the development of sustainable colorant alternatives for printing and creative applications while supporting environmentally responsible biomass management strategies.

Although invasive alien plant species have increasingly been explored as alternative sources of natural colorants, most prior studies have focused on textile dyeing or on extracting bioactive compounds rather than developing solid pigments and inks for printing. For example, invasive species such as Impatiens glandulifera and Japanese knotweed (Fallopia japonica) have been investigated as sources of natural dyes for screen-printing on paper and textiles, demonstrating feasibility at the dye stage and in printing trials [20]. Similarly, invasive plants such as Lantana camara have been reported as dye sources with evaluated fastness on cotton [21], reinforcing the broader potential of invasive biomass as a colorant feedstock. In parallel, our previous work and related studies on the Umia River basin species [19] have examined how extraction conditions influence phenolics/anthocyanins recovery from Eucalyptus globulus, Tradescantia fluminensis, and Arundo donax, but without advancing toward pigment solidification and ink performance. Building on this context, the novelty of the present study is the end-to-end workflow integration that converts IAS biomass into functional printing materials—combining a comparative assessment of five aqueous solid–liquid extraction methods, lake pigment precipitation, oil-based ink formulation, and microscopy/SEM-based printing performance evaluation—thereby moving beyond dye feasibility toward pigment/ink applicability in printmaking and future scale-up in craft and non-industrial contexts.

2. Materials and Methods

2.1. Selected Species and Area of Study

Plant biomass was collected from a riparian forest ecosystem along the Umia River (Galicia, northwestern Spain), within a 2 km stretch located between the coordinates 42°31′28.4808″ N, 8°45′32.6628″ W and 42°31′28.4844″ N, 8°45′32.6736″ W. The area is characterized by a high presence of invasive and non-native plant species and is subject to periodic vegetation removal as part of ecosystem management practices.

Sampling was conducted between May and November 2023. The collected plant species included Arundo donax, Phytolacca americana, Tradescantia fluminensis, and Eucalyptus globulus. Different anatomical parts were selected depending on the species: stems and leaves for the herbaceous species, and bark and leaves for Eucalyptus globulus. Fruits were not included in the study.

After collection, the biomass was air-dried at ambient temperature (20–22 °C) and stored under dry conditions in the Wood Chemistry Laboratory of the Forestry Engineering School, University of Vigo (Pontevedra campus). Prior to extraction, dried samples were manually cut into pieces of approximately 2.0 cm to increase the solid–liquid contact surface during extraction.

2.1.1. Arundo donax

Arundo donax is a perennial herbaceous macrophyte belonging to the Poaceae family, commonly found in riparian and wetland ecosystems. The species exhibits vigorous vegetative propagation through rhizomes, which contributes to its high biomass production and rapid regrowth capacity, features that are relevant for its consideration as a raw material source [2,22].

Due to its extensive rhizome system, which can reach several meters in depth, Arundo donax shows strong resilience and persistence after mechanical removal, resulting in the generation of large amounts of residual biomass during management actions [22]. Although originally introduced for agricultural purposes, the species is currently recognized as one of the most problematic invasive plants in Europe, a factor that supports its availability as waste material for valorization processes [2].

From a material and chemical perspective, Arundo donax biomass has been reported to contain a wide range of biocompounds, including alkaloids, sterols, triterpenes, phenolic compounds, lignin, and carotenoids, distributed across leaves, stems, rhizomes, and roots [23]. These compounds are associated with antioxidant activity and chromatic properties, making the species suitable for studies focused on dye and pigment extraction.

In addition to its invasive behavior, Arundo donax has been investigated for its potential applications in ethnopharmacology, construction, papermaking, and bio-based materials, highlighting its versatility as a lignocellulosic resource [19,24,25,26]. In the present study, stems and leaves of Arundo donax were selected as the plant material for solid–liquid extraction experiments.

2.1.2. Phytolacca americana

Phytolacca americana is a perennial herbaceous plant characterized by a robust root system and vigorous vegetative growth. The species is widely distributed in Mediterranean and Atlantic regions and is classified as an invasive alien species in Spain and under European regulations, which contributes to its high availability as residual biomass following control and removal actions [27,28].

From a material perspective, Phytolacca americana is of particular interest due to the presence of bioactive and chromophoric compounds in its leaves and fruits. Previous studies have reported that its biomass contains phenolic compounds, flavonoids, saponins, betalains, and carotenoids, which are responsible for its characteristic coloration and antioxidant properties [29]. These compounds make the species suitable for investigations focused on natural dye and pigment extraction.

Although Phytolacca americana is known for its toxicity, especially in fruits and roots, this characteristic does not interfere with the extraction procedures applied in this study, as only vegetative parts were handled and processed. The toxicity of the species has, however, been historically associated with its limited use despite its intense coloration [27].

In addition to its invasive behavior, Phytolacca americana has been previously studied for its pharmacological potential, including antibacterial, antiviral, antifungal, and antioxidant activities, further supporting the relevance of its chemical composition [29]. In the present work, stems and leaves of Phytolacca americana were selected as the raw material for solid–liquid extraction experiments, while fruits were intentionally excluded.

2.1.3. Tradescantia fluminensis

Tradescantia fluminensis Vell. is a perennial herbaceous plant belonging to the Commelinaceae family, characterized by rapid horizontal growth and a high adaptability to humid and shaded environments. This species forms dense vegetative mats that limit light availability and space for native flora, which results in the generation of substantial amounts of residual biomass during management and removal activities, particularly in riparian ecosystems [30,31].

From a material standpoint, Tradescantia fluminensis presents soft, water-rich tissues with a relatively low lignin content compared to woody species, which may influence extraction efficiency and pigment stability. Previous studies have demonstrated that biomass from this species contains a wide range of bioactive compounds, including coumarins, alkaloids, saponins, flavonoids, phenolic compounds, tannins, steroids, and terpenoids, many of which exhibit antioxidant properties relevant to dye and pigment extraction processes [4,32].

The invasive behavior and fast vegetative propagation of Tradescantia fluminensis facilitate its widespread availability as waste biomass, while its chemical composition supports its potential reuse in sustainable material applications. Earlier research has reported the presence of antioxidant and phenolic compounds in extracts obtained from this species, reinforcing its suitability for further investigation as a source of natural colorants [4,19].

In the present study, aerial vegetative parts (stems and leaves) of Tradescantia fluminensis were selected as the plant material for solid–liquid extraction experiments.

2.1.4. Eucalyptus globulus

Eucalyptus globulus Labill. is a perennial arboreal species belonging to the Myrtaceae family and is widely cultivated in northern Spain due to its fast growth and industrial relevance, particularly in the pulp and paper sector. Although it is not officially classified as an invasive alien species in Spain, its extensive monoculture plantations and associated ecological impacts have generated increasing concern, especially in regions such as Galicia [1,33,34].

From a material perspective, Eucalyptus globulus differs substantially from the herbaceous species included in this work due to its woody structure, higher lignocellulosic content, and chemically complex secondary metabolite profile. Phytochemical characterization reported in previous studies has identified more than 37 constituents distributed across leaves, bark, aerial parts, and fruits [35]. These include a wide range of phenolic compounds such as quercetin, luteolin, kaempferol, isorhamnetin, phloretin, and chlorogenic acid, as well as polyphenols and tannins, particularly concentrated in the bark [9,19,35].

In addition to phenolics, Eucalyptus globulus biomass contains other bioactive compounds such as eucalyptol, D-limonene, cardiac glycosides, steroids, and alkaloids, which contribute to its antioxidant properties and may influence extraction behavior and pigment formation [35]. The high tannin and polyphenol content of bark and leaves is particularly relevant for this study, as these compounds are known to be associated with brown and ochre chromatic tones and increased pigment stability.

Industrial processing of Eucalyptus globulus primarily exploits the woody stem, while bark and leaves are typically discarded as waste and frequently subjected to burning. Previous studies have demonstrated that these residual fractions constitute a valuable source of antioxidants, phenolics, and tannins, supporting their reuse in sustainable material and pigment applications [19,36,37].

In the present study, bark and leaves of Eucalyptus globulus were selected as the raw material for solid–liquid extraction experiments, allowing for the assessment of pigment extraction from woody biomass and comparison with herbaceous plant sources.

2.2. Solid–Liquid Extraction Methods

Solid–liquid extraction was employed to recover plant-derived colorants from the selected biomass. In this study, a clear distinction is made between dyes and pigments based on their solubility and behavior in the application medium. Dyes are defined as organic colorants that are soluble in the extraction solvent, whereas pigments are insoluble materials that must be dispersed in a binding medium for application [8,38]. As most plant-derived colorants are initially obtained as soluble dyes, an additional solidification step is required to convert them into pigments suitable for printing and ink formulation [17].

Five solid–liquid extraction methods were selected to obtain liquid dyes from invasive and non-native plant biomass under different thermal and mechanical conditions: infusion, infusion with heat, thermal agitation, Soxhlet extraction, and ultrasonic-assisted extraction. These methods were chosen based on their common use in botanical dye extraction and plant-derived compound recovery, as well as their different operational intensities [36].

The selection of multiple extraction methods enables a comparative evaluation of extraction efficiency, color preservation, and suitability for subsequent pigment formation. In particular, extraction temperature and duration were considered critical parameters, as excessive thermal or oxidative conditions may degrade chromophoric compounds and alter the chromatic properties of the resulting pigments [8,36].

To ensure comparability among extraction methods, all experiments were performed using the same solid-to-liquid ratio, with 20 g of dried plant material and 250 mL of solvent per extraction. Distilled water was selected as the extraction solvent in all cases to avoid the use of organic solvents or additional chemical agents, in line with sustainability-oriented objectives [38]. Extraction time and temperature were defined according to the operational characteristics of each method and are summarized in

Table 2. Relation of solid–liquid extraction methods used and time and temperature of experiments.

Solid–Liquid Extraction Methods	Time	Temperature
Infusion Extraction	48 h	20–22 °C
Infusion with heat Extraction	1 h	100 °C
Thermal Agitator Extraction	8 h	40 °C
Soxhlet Extraction	2.5 h	100 °C
Ultrasonic-Assisted Extraction	40 min	70 °C

.

After extraction, the liquid dyes were filtered to remove solid residues and stored for subsequent conversion into solid pigments via a lake pigment precipitation process, as described in Section 2.3.

2.2.1. Infusion Solid–Liquid Extraction

Infusion extraction was performed as a low-energy, room-temperature solid–liquid extraction method commonly used for the recovery of plant-derived dyes. For each extraction, 20 g of dried and cut plant material was placed in a glass container and mixed with 250 mL of distilled water, maintaining a constant solid-to-liquid ratio.

The mixture was left to stand for 48 h at ambient temperature (20–22 °C) without the application of external heat or mechanical agitation. During this period, soluble colorant compounds were gradually released from the plant biomass into the aqueous medium. After the extraction time, the liquid phase was separated from the solid residue by filtration using standard laboratory filter paper. The resulting liquid dye extract was collected and stored for subsequent processing.

This extraction method was selected to evaluate the efficiency and color preservation of a mild extraction process, minimizing thermal degradation and oxidation of chromophoric compounds.

2.2.2. Infusion with Heat Solid–Liquid Extraction

Infusion with heat extraction was carried out to evaluate the effect of elevated temperature on the release of plant-derived dyes and colorant compounds. For each extraction, 20 g of dried and cut plant material was placed in a glass container and mixed with 250 mL of distilled water, maintaining the same solid-to-liquid ratio used in the other extraction methods.

Heat was applied to the container using a domestic heating plate, bringing the aqueous mixture to boiling temperature (approximately 100 °C). The extraction was maintained at boiling conditions for 1 h without mechanical agitation. After completion of the extraction period, the mixture was allowed to cool to room temperature.

The liquid phase was then separated from the solid biomass by filtration using standard laboratory filter paper. The resulting liquid dye extract was collected and stored for subsequent conversion into solid pigments.

This method was selected to assess the influence of thermal energy on extraction efficiency and dye stability under conditions that remain accessible for low-technology and small-scale applications.

2.2.3. Thermal Agitator Solid–Liquid Extraction

Thermal agitation extraction was performed to combine moderate heating with mechanical agitation in order to enhance mass transfer between the solid plant material and the solvent. For each extraction, 20 g of dried and cut plant material was mixed with 250 mL of distilled water, maintaining a constant solid-to-liquid ratio.

The extraction was conducted using a benchtop shaking incubator (Corning LSE, model 6791 EU;, Corning Incorporated, Corning, NY, USA). The temperature was set to 40 °C, and the samples were subjected to orbital shaking for a total extraction time of 8 h, divided into two consecutive periods of 4 h. The shaking orbit of the equipment was 19 mm, with a temperature accuracy of ±0.5 °C. Relative humidity during operation was maintained at approximately 85%, according to the equipment specifications.

After completion of the extraction process, the liquid phase was separated from the solid residue by filtration using standard laboratory filter paper. The resulting liquid dye extract was collected and stored for subsequent processing into solid pigments.

This method was selected to evaluate the effect of prolonged moderate temperature combined with mechanical agitation on dye extraction, while minimizing thermal degradation associated with higher-temperature techniques.

2.2.4. Soxhlet Solid–Liquid Extraction

Soxhlet extraction was employed as a conventional high-temperature solid–liquid extraction method to evaluate maximum extraction yield under continuous solvent reflux conditions. For each extraction, 20 g of dried and cut plant material was placed in a cellulose extraction thimble and introduced into a Soxhlet apparatus.

The extraction was carried out using a heating mantle (Nahita Blue Series 655; Auxilab S.L., Beriáin, Navarra, Spain) connected to a round-bottom flask containing 250 mL of distilled water as solvent. The system was operated at the boiling point of water (approximately 100 °C). A total of five extraction cycles were performed, each lasting 30 min, resulting in a total extraction time of 2.5 h.

During the extraction process, the solvent was continuously refluxed, allowing repeated contact between the solvent and the plant biomass. After completion of the extraction cycles, the apparatus was allowed to cool to room temperature. The liquid extract was then collected and filtered to remove any remaining solid particles. The resulting aqueous dye extract was stored for subsequent conversion into solid pigments. This method was selected to assess extraction efficiency under intensive thermal conditions and to serve as a reference for comparison with milder extraction techniques.

2.2.5. Ultrasonic Extraction Solid–Liquid Extraction

Ultrasonic-assisted extraction was applied to evaluate the effect of acoustic cavitation on the release of plant-derived dyes and colorant compounds, allowing reduced extraction time compared to conventional techniques. For each extraction, 20 g of dried and cut plant material was mixed with 250 mL of distilled water, maintaining the same solid-to-liquid ratio used in the other extraction methods.

The extraction was performed using an ultrasonic bath (Bandelin Sonorex Super RK 102 H; BANDELIN electronic GmbH & Co. KG, Berlin, Germany) operating at a frequency of 35 kHz. The extraction temperature was set to 70 °C, with a maximum operating temperature of 80 °C according to the equipment specifications. Ultrasonic treatment was applied for a total extraction time of 40 min.

After completion of the ultrasonic treatment, the mixture was allowed to cool to room temperature. The liquid phase was then separated from the solid residue by filtration using standard laboratory filter paper. The resulting aqueous dye extract was collected and stored for subsequent conversion into solid pigments.

This method was selected to assess the efficiency of ultrasound-assisted extraction in recovering colorant compounds under reduced processing time and moderate thermal conditions.

2.3. Solidification Method: Lake Pigment Formation

Following solid–liquid extraction, the obtained plant-derived colorants were present in the form of soluble aqueous dyes. In order to obtain solid pigments suitable for ink formulation and printing applications, the liquid dyes were converted into insoluble pigments through a lake pigment precipitation process. This method is based on the formation of metal–dye complexes, resulting in the insolubilization of organic colorants [8,17,18]. A schematic representation of the dye-to-pigment conversion process is shown in Figure 1.

Lake pigment formation in this study is based on established aluminum–phenolic coordination chemistry reported in historical and modern pigment literature. Upon dissolution of aluminum potassium sulfate dodecahydrate (KAl(SO₄)₂·12H₂O, alum) in water, Al³⁺ ions are released and rapidly form hydrated hexaaquo complexes, [Al(H₂O)₆]³⁺, as described in aqueous aluminum coordination chemistry [13,17].

Phenolic compounds, flavonoids, and anthocyanin-related structures present in the plant extracts contain hydroxyl and carbonyl functional groups capable of acting as ligands. In particular, catechol-type dihydroxyl groups and hydroxyl–carbonyl arrangements in flavonoid structures are known to chelate trivalent metal ions, including Al³⁺ [11,13,17]. Aluminum ions behave as Lewis acids and coordinate with electron-donating oxygen atoms from deprotonated phenolic groups.

The gradual addition of sodium bicarbonate increases the pH of the solution, promoting partial deprotonation of phenolic –OH groups and enhancing their coordination capacity. This pH adjustment also favors hydrolysis of aluminum species, facilitating the formation of insoluble aluminum–organic complexes. The resulting Al³⁺–dye chelates exhibit reduced solubility, leading to precipitation of a metal–organic complex commonly referred to as a lake pigment [8,17,39,40].

Although the precise stoichiometry and coordination geometry were not determined in this work, the formation of aluminum–phenolic complexes is consistent with mechanisms previously reported for natural lake pigments and metal-mordanted dyes [8,13,39,41,42].

In a practical level, lake pigment formation was carried out using the mentioned aluminum potassium sulfate dodecahydrate (KAl(SO₄)₂·12H₂O) as the metal salt and sodium bicarbonate (NaHCO₃) as the alkaline agent to induce precipitation. Preliminary tests were conducted to evaluate the influence of reagent concentration and dye volume on pigment formation and color quality (

Table 3. Performed tests with different ratio content for lake pigment extraction.

Test	Dye Content (mL)	Aluminum Potassium Sulfate	Sodium Bicarbonate (g)
1	200	5	2.5
2	200	10	5
3	200	15	10
4	100	5	2.5
5	100	10	5
6	100	15	10

). Based on these tests, two conditions (tests 3 and 5) showed improved pigment formation and color retention, with test 5 selected as the most efficient condition due to reduced dye volume and satisfactory chromatic results.

For lake pigment preparation under the selected conditions, 100 mL of liquid dye extract was diluted with 200 mL of distilled water under continuous stirring. Subsequently, 10 g of aluminum potassium sulfate was added to the solution and allowed to dissolve completely. Sodium bicarbonate (5 g) was then gradually added to the mixture to promote precipitation of the metal–dye complex. The formation of a solid precipitate indicated the conversion of the soluble dye into an insoluble lake pigment.

After completion of the precipitation reaction, the suspension was allowed to decant at ambient temperature (Figure 2). The solid pigment was separated from the supernatant by filtration using standard laboratory filter paper. The collected pigment was then dried at ambient temperature for 48–72 h until complete dehydration was achieved.

The dried pigments were manually ground prior to further characterization and ink formulation. The lake pigment preparation process was applied to all dye extracts selected for pigment production.

2.4. Pigment Grinding and Ink Formulation

After drying, the obtained lake pigments were manually ground to reduce particle size prior to ink preparation. As industrial printing inks typically contain pigment particles below the micron scale, particle size reduction was performed manually using a knurling tool, acknowledging that laboratory-scale manual processing does not achieve industrial milling standards [10]. Each pigment sample was ground for 10 min, repeated twice.

Oil-based (greasy) inks were formulated by mixing ground pigments with linseed oil as binder. For each formulation, 3.0 g of dry pigment was combined with 1.5 g of linseed oil. The mixture was manually homogenized using the knurling tool until a uniform paste suitable for manual application was obtained. No additional solvents, additives, or drying agents were incorporated into the ink formulations.

The resulting inks were subsequently used for particle size measurement and printing tests as described in the following sections.

2.5. Optical Analysis and Properties Determination of Pigments and Greasy-Inks

Optical and morphological properties of the obtained pigments and formulated greasy inks were evaluated to assess color appearance, particle distribution, and ink interaction with paper substrates. The analyzed properties included optical stability, coloring capacity, grain distribution, and ink penetration/absorption behavior on the substrate surface.

Printing tests were carried out by manual application of the formulated inks onto commercial printmaking paper. The printed samples were subsequently examined by microscopy to evaluate ink distribution and penetration into the paper fiber network.

Scanning electron microscopy (SEM) was employed to visualize fiber structure, ink absorption, and pigment particle distribution after printing. SEM observations were performed using a JEOL 6100 electron microscope (JEOL Ltd., Akishima, Tokyo, Japan). In addition, surface appearance and color characteristics were documented using an optical stereomicroscope (Nikon SZM 1500; Nikon Corporation, Tokyo, Japan). To support visual comparison of color and surface distribution, printed samples were also digitized using a flatbed scanner (Epson Perfection V800; Seiko Epson Corporation, Suwa, Nagano, Japan). Images obtained from SEM, optical microscopy, and scanning were analyzed to compare pigment/ink behavior and surface coverage among the different plant sources and extraction conditions.

2.6. Statistical Analysis

All experiments were performed in triplicate. The reported values correspond to the arithmetic mean of three independent measurements. Data processing and basic statistical analysis were carried out to evaluate the reproducibility of the experimental procedures.

Results are presented as mean values, and variability among replicates was considered in the interpretation of the data. No advanced statistical tests were applied, as the study focuses on comparative and exploratory assessment of extraction methods, pigment formation, and ink behavior within an specific case of study.

3. Results

3.1. Extraction Yield and Visual Characteristics of Liquid Dyes

The extraction yield obtained from the different plant species showed notable variation depending on the extraction method applied. Among the evaluated techniques, Soxhlet extraction consistently resulted in the highest extraction yields, reaching values of up to 30.5%, whereas lower yields were generally obtained using milder extraction methods such as infusion and ultrasonic-assisted extraction.

However, differences in extraction yield were accompanied by noticeable variations in the visual characteristics of the resulting liquid dyes. Although Soxhlet extraction produced higher yields, the corresponding extracts frequently exhibited darker color tones and, in some cases, signs of oxidation or color degradation. In contrast, extracts obtained by infusion with heat displayed more homogeneous coloration and better visual stability.

Visual differences were also observed among the plant species analyzed. Extracts derived from Eucalyptus globulus typically showed darker brown tonalities, while those obtained from Arundo donax, Tradescantia fluminensis, and Phytolacca americana presented lighter yellow to greyish hues, depending on the extraction method employed.

Representative images of the liquid dyes obtained from the different extraction methods are presented in Figure 3. Also, in comparison with previous results [19], even it is possible to find a correlation between plant species and type of method extraction yield, for the present study it has been decided to perform the comparison by using only Eucalyptus bark as biomass to select the most suitable solid–liquid extraction method.

Based on these observations, infusion with heat was selected for subsequent pigment production, as it provided a balance between extraction yield and color stability.

3.2. Color, Pigment and Ink Properties

Based on the extraction results described in Section 3.1, dyes obtained by infusion with heat were selected for solid pigment formation due to their superior preservation of color properties and reduced oxidation. These dyes were subsequently solidified through the lake pigment process, yielding pigments with distinct chromatic characteristics depending on the botanical source.

As shown in Figure 4 and Figure 5, pigments obtained from Arundo donax exhibited strong coloring capacity, resulting in bright yellow lake pigments. These pigments maintained high color intensity after drying and demonstrated visual similarities to lemon yellow pigments, also known as yellow ultramarine or barium yellow (BaCrO₄). Synthetic lemon yellow pigments are traditionally produced through high-temperature reactions between potassium dichromate and barium carbonate [39].

Pigments derived from Tradescantia fluminensis displayed a slightly bluish tone when fully dried, as illustrated in Figure 6 and Figure 7. This coloration may be associated with a combination of graphite-based compounds and aerinite-like pigments. Graphite, also referred to as plumbago or black lead, is a naturally occurring mineral [40,41], while the bluish hue observed could indicate the presence of small quantities of aerinite diluted within the pigment matrix. Additionally, this pigment may serve as a potential substitute for slate gray powder, which, although not synthetic, exhibits similar chromtic variations.

Figure 8 and Figure 9 present pigments obtained from Phytolacca americana, characterized by a brownish–yellow tone that remained intense in the wet state but decreased in intensity after complete drying. Visual similarities were observed with chrome yellow pigments (PbCrO₄) [40], also known as primrose chrome or lemon chrome, as well as with cadmium yellow pigments (CdS). Chrome yellow pigments are typically prepared from soluble lead salts combined with potassium or sodium chromate, whereas cadmium yellow pigments are obtained through reactions involving cadmium salts and hydrogen sulfide or alkali sulfides [40,41].

The pigments obtained from Eucalyptus globulus are shown in Figure 10 and Figure 11. The resulting brown tonalities are consistent with the high tannin content reported for both bark and leaves of this species [2,9]. The pigment visually resembles natural umber pigments, which are traditionally sourced from mineral deposits in southern Europe and require drying and grinding prior to use [40,41].

After the tests were performed, it was observed that the use of leaves or stem material did not significantly affect the coloring capacity of the pigments in any of the studied species.

Due to the lack of spectrophotometric equipment for direct color measurements, an interactive online color map was used to estimate the CIE-Lab coordinates of the formulated inks. Color comparisons were carried out visually against the reference map, and the corresponding CIE-Lab values are presented in

Table 4. Observed CIE-Lab information from elaborated inks from extracted pigments.

Colour	Raw Matter	CIE-Lab Data	RGB Data
	Phytolacca americana	L: 81	R: 240
		A: 15.18	G: 218
		B: 56.63	B: 107
	Arundo donax	L: 94	R: 247
		A: −7.25	G: 234
		B: 38.58	B: 141
	Eucalyptus globulus	L: 80	R: 223
		A: 13.85	G: 191
		B: 51.56	B: 114
	Tradescantia fluminensis	L: 88	R: 237
		A: 0.67	G: 217
		B: 25.54	B: 144

.

Due to the absence of spectrophotometric instrumentation at the time of the study, color evaluation was performed through visual comparison using a standardized digital CIE-Lab and RGB reference chart. The reported values therefore represent approximate estimations intended to provide comparative chromatic positioning rather than precise quantitative colorimetry.

The obtained CIE-Lab coordinates should be interpreted as indicative of relative hue differences among the inks rather than absolute chromatic measurements. The approach allowed preliminary comparison of tonal tendencies among pigments derived from different plant species but does not substitute for instrumental colorimetric characterization.

Future work will include UV–Vis absorbance spectroscopy of liquid extracts, reflectance spectrophotometry of solid pigments, and ΔE-based color difference analysis to provide objective chromatic quantification and stability assessment.

According to previous studies [19], Tradescantia fluminensis, Arundo donax, and Eucalyptus globulus are rich sources of antioxidants, anthocyanins, and total phenolic compounds. In particular, Eucalyptus globulus exhibits average values of 2.67 ± 0.52 mg CC/g of anthocyanins and 49.72 ± 16.58 mg CC/g of total phenolics, whereas Tradescantia fluminensis shows considerably lower anthocyanin concentrations (0.25 ± 0.11 mg CC/g) and total phenolic content (7.31 ± 1.86 mg CC/g). These compositional differences are reflected in the observed color variations among the pigments. While Eucalyptus pigments display darker brown tones associated with tannin-rich compositions, Tradescantia pigments exhibit lighter and softer hues.

The formulated inks obtained from the extracted pigments are shown in Figure 12 after mixing with linseed oil using a hand knurling tool.

Although the selected reagent ratios produced satisfactory pigment formation under laboratory-scale conditions, lake pigment precipitation is inherently sensitive to physicochemical parameters such as pH, ionic strength, temperature, and addition rate of the alkaline agent. In the present study, pH was not continuously monitored during precipitation, and reaction kinetics were not systematically evaluated. Controlled pH regulation and kinetic analysis would enable improved reproducibility and optimization of particle size and chromatic intensity. Furthermore, exploration of alternative metal salts, such as Fe³⁺ or Ca²⁺, may allow modulation of pigment hue and stability, as reported in traditional mordant-based pigment systems [13,17]. These aspects represent relevant directions for future process refinement and scalability assessment.

Pigment particle sizes were evaluated in both powdered and ink forms. Although the obtained particle sizes remain above industrial standards, the pigments demonstrated adequate suitability in terms of color performance and extraction efficiency. Printing tests were conducted on Hahnemühle paper, and ink behavior on the paper surface was evaluated using SEM imaging. As shown in Figure 13, a comparison between commercial ink and Arundo donax ink highlights differences in particle distribution within the paper fiber network.

Pigment particle size was determined through SEM image analysis by measuring representative particle diameters from three independent observations per sample. The reported values correspond to measured particle dimensions rather than full granulometric distributions. Therefore, they provide an estimation of particle scale rather than statistical descriptors such as D10, D50, or D90 values.

As summarized in

Table 5. Relation of pigment particle size depending on raw matter used.

Raw Matter	Pigment Article Size (μm)
	Min	Medium	Max
Arundo donax	1.204	3.901	7.143
Eucalyptus globulus	1.346	2.100	3.041
Phytolacca americana	5.000	8.347	11.999
Tradescantia fluminensis	3.093	4.242	5.257

, Eucalyptus globulus pigments exhibited the smallest observed average particle dimensions (approximately 2–3 µm), followed by Arundo donax (approximately 3–4 µm). Larger particles were observed for Tradescantia fluminensis (approximately 4–5 µm) and Phytolacca americana (up to 8–12 µm). These differences may be related to variations in organic matrix composition, precipitation dynamics, and manual grinding efficiency.

For comparison, industrial printing pigments typically exhibit submicron to low-micron particle sizes (commonly <1–2 µm) to ensure optimal dispersion, opacity, and surface smoothness. The particle sizes obtained in this study remain above industrial standards, primarily due to manual grinding procedures and the absence of mechanical milling. Implementation of ball milling or high-shear dispersion techniques would likely reduce particle size distribution and improve homogeneity, thereby enhancing ink rheology and covering power. Consequently, the present results should be interpreted as laboratory-scale feasibility outcomes rather than optimized industrial formulations.

3.3. Chemical Composition

Chemical composition analyses were conducted at the CACTI (Centro de Apoio Científico e Tecnolóxico á Investigación) facilities of the University of Vigo. Pigments were previously mixed with linseed oil to formulate greasy inks, which were subsequently analyzed at the CACTI laboratory in Ourense.

Spectrophotometric determination of total phenolic compounds and anthocyanin content was carried out using the Folin–Ciocalteu method for total phenolics and the acid hydrolysis in butanol method for anthocyanins. Previous studies have demonstrated high antioxidant content in the condensed extraction media obtained from the selected plant species [19], supporting the relevance of chemical characterization of the resulting inks.

The elemental chemical composition of the formulated inks was analyzed using a JEOL 6100 scanning electron microscope equipped for compositional analysis. SEM–EDS analysis allowed the identification and comparison of the main elemental components present in the inks derived from the different plant species. The most relevant detected elements included carbon (C), oxygen (O), aluminum (Al), sulfur (S), sodium (Na), potassium (K), calcium (Ca), and silicon (Si).

Figure 14, Figure 15, Figure 16 and Figure 17 present representative SEM–EDS spectra corresponding to the inks formulated from Arundo donax, Eucalyptus globulus, Tradescantia fluminensis, and Phytolacca americana, respectively. In all cases, carbon and oxygen were identified as the predominant elements, consistent with the organic nature of both the pigment compounds and the linseed oil binder. Aluminum was also detected as a major component, associated with the aluminum-based lake pigment formation process. Minor contributions of potassium, sodium, sulfur, calcium, and silicon were observed depending on the botanical source.

A comparative representation of the elemental composition of the inks is presented in Figure 18 and Figure 19. Figure 18 summarizes the relative carbon and oxygen content across all elaborated inks obtained from the selected plant species. Figure 19 highlights the secondary elemental composition, including calcium, silicon, aluminum, sodium, and sulfur, allowing comparison among inks derived from different invasive alien species.

4. Discussion

The chromatic behavior of the obtained pigments is closely linked to the chemical composition of the selected invasive and non-native plant species. The results reveal a consistent relationship between total phenolic and anthocyanin content and the observed tonal characteristics of the resulting pigments and inks, in agreement with previous reports [9,19]. Bark, stem, and leaf tissues from the studied species contain substantial concentrations of antioxidant compounds, which directly influence chromophore availability during extraction and subsequent pigment formation.

The predominance of yellow, ochre, and brown tonalities across the analyzed samples aligns with the known chromatic properties of flavonoid- and tannin-rich matrices [34,41]. Woody species, characterized by higher tannin and phenolic content, generated darker and more complex brown hues, whereas the herbaceous species Tradescantia fluminensis produced lighter and less saturated tones. These differences highlight the role of plant tissue composition and secondary metabolite profiles in determining pigment color expression.

The comparative evaluation of extraction methods demonstrated a trade-off between extraction yield, chromatic preservation, and process intensity. Although Soxhlet extraction achieved the highest quantitative yields (up to 30.5%), the prolonged reflux conditions promoted oxidative darkening and degradation of chromophoric compounds. Additionally, continuous boiling and solvent recirculation imply greater thermal energy demand compared to infusion-based techniques. In contrast, infusion with heat achieved satisfactory extraction efficiency under shorter thermal exposure without reflux, reducing both oxidative effects and process intensity.

From both chromatic and operational perspectives, infusion with heat provided the most balanced compromise between extraction efficiency, color preservation, and energy demand. For printing applications, where hue clarity and tonal consistency are critical, preservation of chromatic integrity proved more relevant than maximization of extraction yield. While a full Life Cycle Assessment was beyond the scope of this study, qualitative comparison suggests that milder aqueous extraction methods may better align with circular economy objectives than more energy-intensive approaches. All extractions were performed using water as solvent, minimizing environmental burden, and lake pigment formation relied on aluminum potassium sulfate and sodium bicarbonate, reagents commonly employed in traditional pigment systems.

The lake pigment formation process further revealed the sensitivity of pigment characteristics to chemical and procedural parameters. Ink concentration alone did not determine final color intensity; rather, precipitation dynamics during the aluminum–dye complexation stage played a decisive role. Excessive addition of aluminum salt or alkaline agent resulted in desaturated or whitish pigments, while variations in decantation time and ambient temperature influenced both pigment yield and particle size. These findings underscore the importance of controlled precipitation parameters for reproducibility and future process optimization.

When incorporated into linseed oil binders, the pigments exhibited enhanced color depth and saturation relative to their powdered state. SEM observations confirmed adequate pigment distribution and penetration within the paper fiber network, demonstrating functional applicability in manual printmaking. However, the laboratory-prepared inks were softer and less dense than commercial formulations, reflecting the limitations of manual grinding and the absence of rheological optimization. In particular, pigments derived from Tradescantia fluminensis displayed limited coloring capacity, with binder interaction partially masking bluish tonal components. Quantitative rheological characterization, adhesion testing, opacity measurements, abrasion resistance evaluation, and penetration depth quantification were not performed and will be necessary to benchmark IAS-derived inks against industrial printing standards.

Long-term pigment durability and lightfastness were not experimentally assessed in the present feasibility study. Nevertheless, conversion of soluble botanical dyes into aluminum-based lake pigments is known to enhance physicochemical stability relative to unmodified extracts, as metal–ligand coordination reduces dye solubility and molecular mobility [8,17]. While the antioxidant and phenolic content of the selected species may contribute to chromatic robustness, actual long-term stability depends on chromophore structure and environmental exposure conditions. Instrumental colorimetric characterization was also not conducted; CIE-Lab and RGB values were estimated for comparative positioning only and should not be interpreted as precise quantitative measurements. Future work should incorporate reflectance spectrophotometry, UV–Vis analysis, ΔE-based color difference calculations, and controlled aging experiments to objectively evaluate chromatic stability and durability.

The objective of this study was to assess the technical feasibility of transforming IAS biomass into lake pigments and oil-based inks under laboratory-scale conditions, with emphasis on manual printmaking applications. Industrial scalability was not within the scope of the present work. Translation to offset, flexographic, or other commercial printing systems would require additional optimization, particularly in pigment particle size reduction, dispersion stability, and rheological control. Although the use of water as solvent and relatively simple precipitation chemistry suggests potential scalability, standardized pH control, precipitation kinetics optimization, and mechanical milling would be necessary to achieve industrially consistent granulometry and ink performance. Consequently, the present work establishes proof of concept, while future research will determine the practical and economic feasibility of large-scale implementation.

5. Conclusions

The experimental results confirm the viability of using vegetal residues from invasive alien species (IAS)—specifically Arundo donax, Phytolacca americana, Tradescantia fluminensis, and Eucalyptus globulus—as raw materials for the development of greasy inks suitable for hand-printing techniques. The study demonstrates that the valorization of IAS biomass represents a feasible strategy for integrating environmental sustainability into creative and printing practices.

Among the extraction methods evaluated, infusion with heat was identified as the most effective approach for preserving dye color integrity, despite Soxhlet extraction providing higher overall extraction efficiency. The gentler thermal conditions of the infusion method limited dye oxidation and resulted in improved performance during the lake pigment formation process, particularly in terms of color retention and tonal clarity.

Significant differences in chromatic behavior were observed among the studied species. Pigments derived from Phytolacca americana and Arundo donax exhibited higher coloring capacity, generating intense and bright yellow hues with variable tonalities. In contrast, Eucalyptus globulus produced darker pigments associated with its high tannin content, while Tradescantia fluminensis yielded lighter and softer brown tones, especially when dispersed in linseed oil, although with lower overall pigment strength.

Particle size analysis revealed notable variation depending on the botanical source. The results indicate that the use of mechanical grinding methods, rather than manual processing, could enhance particle size uniformity and improve ink performance, constituting a relevant line for future investigation.

In addition, further research is required to evaluate the chemical residues generated during the lake pigment formation process. Although complete zero-waste processing cannot be conclusively established, preliminary observations suggest that the residual chemical content after filtration is limited. This opens opportunities for future studies focused on the characterization of remaining dye compounds and the optimization of the environmental impact of the pigment production process.

Overall, the reintegration of IAS-derived biomass into pigment and ink production systems offers a sustainable alternative for the creative industry, supporting the development of artistic and printing practices aligned with ecological principles and circular economy goals.