Sustainable Dyeing and Functionalization of Knitted Cotton Fabrics with Algae Extracts

Abstract

Algae extracts have emerged as a sustainable and eco-friendly alternative to synthetic dyes and functional additives in the textile industry, particularly for dyeing and functionalizing of cotton fabrics. Herein, two types of water-soluble algae extracts from Arthrospira platensis and Porphyridium cruentum were characterized in terms of thermal, structural, and functional properties and used as dye and/or functional agents. Cotton samples were pre-treated with chitosan and alum mordants and compared with commercially treated cationic cotton. The algae extracts were applied through the exhaust method, with variations in temperature, pH, liquor ratio, temperature rise gradient, and extract percentages. The resulting colours, assessed through CIE L*a*b* coordinates and K/S values using UV–Vis spectroscopy, displayed green and pink coloration, with commercial cationic cotton exhibiting more intense colours. Colour fastness measurements were also performed on functionalized cotton fabrics. The water-based algae extracts and functionalized samples were additionally characterized for functional features, displaying an antioxidant activity exceeding 60% (68.13 ± 3.60 and 60.76 ± 1.18, for A. platensis and P. cruentum, respectively). This work highlights their dual role in providing both aesthetic dyeing and functional enhancement of cotton. By using renewable marine resources and eco-friendly water-based processes, this approach supports the development of greener, more sustainable textile technologies.

1. Introduction

The increasing demand for alternative solutions in the textile industry has driven extensive research into natural dyes and bio-based functional finishes. Conventional synthetic dyes, while offering a wide colour palette and high performance, are associated with significant environmental concerns, including toxic effluents, low biodegradability, and potential risks to human health [1]. As a result, interest has grown in renewable sources of natural pigments that can provide both coloration and added functionality to textiles.

Natural dyes derived from renewable biological sources have re-emerged as viable alternatives due to their biodegradability and lower environmental impact [2,3]. Beyond coloration, many natural colourants also exhibit bioactive properties, including antioxidant, antimicrobial, and UV-protective functions, enabling simultaneously aesthetic and functional enhancement of textiles. However, their commercial application remains limited due to challenges such as low dye uptake, poor reproducibility, and limited fastness to washing and light [2,4,5,6]. Addressing these limitations requires careful optimization of extraction, mordanting, and dyeing parameters, as well as exploring new biological dye sources with richer pigment composition and higher functional potential.

Among these sources, algae have gained particular interest as promising natural pigments and bioactive compounds [6,7,8,9,10]. Algae encompass a diverse group of photosynthetic organisms, including microalgae and macroalgae, that can be cultivated sustainably. As they are a rich source of bio-pigments—including chlorophylls, carotenoids, and phycobiliproteins [6,7], as well as in phenolic compounds and polysaccharides—algae can provide not only vibrant colours but also functionalities [6,7,11,12]. These characteristics make algae extracts appealing not only as natural dyes but also as functional agents capable of adding value to the textile substrates.

Nevertheless, the practical application of algae-derived pigments in textile dyeing is still limited by several challenges. The pigment molecules are often sensitive to temperature, pH, and light exposure, which can lead to degradation or colour shifts during processing [7,9,10,13,14]. Their water solubility and molecular charge also affect dye–fibre interactions, resulting in limited affinity towards natural fibres such as cotton. To overcome these limitations, pre-treatments such as mordanting with metal salts, natural polymers, or chemical fibre modifications (cationization) have been investigated to improve dye uptake and fixation [2,11,14]. Moreover, the optimization of dyeing parameters—including temperature, pH, liquor ratio, and temperature rise gradient—is crucial in achieving a balance between pigment stability, bath exhaustion, and colour strength [13].

Among the diverse species of microalgae, Arthrospira platensis and Porphyridium cruentum stand out for their rich protein and pigment composition, bright colours, biochemical diversity, and multifunctional potential [15,16,17,18]. Arthrospira platensis, a spiral-shaped, filamentous photosynthetic cyanobacteria belonging to the group of blue-green microalgae, is one of the most researched microalgae. This species, commonly known as Spirulina platensis and the source of many studies, has been widely recognized for centuries as a natural source of nutrition and, more recently, for its cosmeceutical potential due to its functional properties, including antioxidant, anti-inflammatory, anti-ageing, anti-wrinkle, and UV-protective effects [16,19]. Arthrospira platensis is particularly rich in proteins [20]—phycobiliproteins, particularly phycocyanin, essential vitamins such as B12 and provitamin A, minerals, and other bioactive compounds [17,19]. Porphyridium cruentum, on the other hand, is a spherical, unicellular red microalga from the rhodophyta phylum [17], characterized for its intense red pigmentation derived from phycoerythrin, a major phycobiliprotein that significantly contributes to its total protein content. It also contains considerable amounts of sulphated polysaccharides, carotenoids, and lipids–mostly unsaturated fatty acids beneficial for cardiovascular health–which provide it with strong antioxidant, antimicrobial, and anti-inflammatory properties [17,21]. Together, the biochemical richness and bioactivity of these two species make them attractive for applications across various sectors, including pharmaceutical, cosmetic, food industries, natural pigment production, and textile natural dyeing and/or functionalization [7,8,13,17,20,22].

Herein, aqueous extracts from Arthrospira platensis and Porphyridium cruentum were explored as dual-purpose agents for the dyeing and functionalization of cotton fabrics. This research investigated how pre-treatments (mordanting with alum and chitosan, with commercial cationization), dyeing parameters (temperature, pH, liquor ratio, temperature rise gradient, extract concentration), and extract composition influence the colour strength, fastness, and antioxidant functionality of the dyed textiles. The functional performance was assessed through antioxidant assays (ABTS and DPPH) and by evaluating colour fastness to washing, water, and perspiration.

By integrating colour performance with bio-functional properties, this work aimed to contribute to the development of more sustainable textile processing approaches while adding value through natural functionalization.

2. Materials and Methods

2.1. Materials and Reagents

Bleached jersey cotton and cationized jersey cotton fabrics (both 150 g/m²) were obtained from Tintex Textiles S.A. (Vila Nova de Cerveira, Viana do Castelo, Portugal); alum (aluminium-potassium sulphate dodecahydrate, 99.5%, AlK(SO₄)₂·12H₂O) and chitosan (MW: 100,000 to 300,000) were acquired from Fisher Scientific, part of Thermo Scientific Chemicals (Porto Salvo, Lisboa, Portugal); Arthrospira platensis (A. platensis) and Porphyiridum cruentum (P. cruentum) microalgae extracts were provided from CeNTI, Portugal. In this work, the term “algae extract” refers to the crude water-soluble extract obtained directly from the algal biomass, which contains a mixture of naturally occurring pigments and other soluble compounds, and was used without further purification.

2.2. Methods

2.2.1. Extracts Characterization

Powder algae extracts were characterized for structural, thermal, and functional properties. Absorbance scans were collected between 190–1100 nm (resolution of 1 nm) using the Shimadzu UV-1800 equipment and high precision quartz cuvettes of type 100-QS (Hellma Analyticals, Hellma GmbH & Co. KG, Müllheim, Germany) for each one of the extracts’ aqueous solutions. Results were plotted as absorbance vs. wavenumber. The chemical composition of extracts was analysed by ATR-FTIR using an IRAffinity-1S, Shimadzu spectrophotometer (Shimadzu Corporation, Kyoto, Japan), coupled with an ATR accessory (diamond crystal). For each sample, a total of 45 scans were performed at a spectral resolution of 2 cm⁻¹, over the wavenumber range of 400–4000 cm⁻¹.

Detection and identification of the compounds was performed using a Thermo Scientific™ Vanquish™ Flex UHPLC system coupled to a Thermo Scientific™ Orbitrap Exploris™ 120 high resolution accurate mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The method was adapted from a previously reported protocol [23] with modifications. The mobile phases consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The flow rate was set at 0.35 mL/min, and the following linear gradient was applied (t in min; %A): 0.0, 95%; 5.4, 85%; 7.9, 80%; 9.0, 80%; 12.6, 70%; 16.2, 50%; 18.0, 5%; 19.8, 95%; 22.0, 95%. The total run time was 27 min. All qualitative data were acquired in data-dependent scanning (DDS) mode. In this mode, the most intense ions detected in the full-scan mass spectrum are automatically selected for subsequent fragmentation, allowing the acquisition of MS/MS spectra and enabling structural elucidation of the compounds. Data were processed using Compound Discoverer™ software (version 3.3, Thermo Scientific™, Waltham, MA, USA), following the workflow previously described by Santos et al. [24]. A final compound list was compiled for critical evaluation.

Thermal gravimetric analysis (TGA) measurements were conducted on an STA 7200 equipment (Hitachi, Ibaraki, Japan). The TGA trace was obtained in the range of 30–600 °C under a nitrogen atmosphere, a flow rate of 200 mL/min, and a temperature rise of 10 °C/min. Results were plotted as the percentage of weight loss vs. temperature. Differential scanning calorimeter (DSC) data were acquired on a Power Compensation Diamond DSC 6000 (PerkinElmer, Waltham, MA, USA). Tests were conducted under a nitrogen atmosphere with a flow rate of 200 mL/min and a heating rate of 10 °C/min. The thermogram was obtained in the range of 0–400 °C. Results were plotted as heat flow vs. temperature.

Finally, the algae extracts were also tested for antioxidant activity (AA) following the ABTS and DPPH methods. The ABTS radical solution was generated by mixing 7.4 mM ABTS with 2.6 mM potassium persulfate (1:1, v/v) and allowing the mixture to stand in a dark at room temperature for 12–16 h. The resulting solution was then diluted with ethanol to obtain an absorbance of 0.70 ± 0.10 at 734 nm. For the DPPH assay, a 0.2 mM solution of DPPH in ethanol was prepared and diluted with ethanol to obtain an absorbance of approximately 0.90 at 515 nm. Briefly, different concentrations of the extracts (0.1–2.5 mg/mL) were prepared to construct inhibition curves. Then, 3000 μL samples of ABTS or DPPH working solution were mixed with 300 μL of algae extract and incubated in the dark for 6 min (ABTS) and 60 min (DPPH). The decrease in absorbance of the samples was measured at 734 (ABTS) and 515 (DPPH) nm using a UV–Vis spectrometer. Radical inhibition was calculated as the reduced percentage, according to Equation (1), where A_control is the absorbance of the control reaction (containing all reagents except for the test sample) and A_sample is the absorbance of the samples at each concentration after the stipulated time. Experiments were performed in triplicate and results are plotted as inhibition percentage vs. extract concentration.

$$\mathrm{R}\mathrm{a}\mathrm{d}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)={\displaystyle \frac{{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}-{\mathrm{A}}_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}}{{\mathrm{A}}_{\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}}\times 100$$

(1)

2.2.2. Pre-Treatment

Two mordants were used: a commercial (alum) and a natural (chitosan). Mordant baths were prepared by using 5.0% wof (weight of fibre) of mordant in water with a liquor ratio of 1:10. Cotton samples were then immersed in the mordant solutions, and the temperature was raised at a rate of 1 °C/min up to 40 °C using the exhaustion machine (AHIBA IR, DataColor, Lucerne, Switzerland). Mordanting process was kept for 40 min before rinsing the samples with tap water and drying at room temperature, as reported by Rahman et al. [5]. The following abbreviations were used: CO—untreated samples; CO_Al—samples mordanted with alum; CO_CS—samples mordanted with chitosan; and CO_cat—cationized samples. The cotton fabric before any process was used as the reference for all performed measurements.

2.2.3. Dyeing

Untreated, pre-mordanted and cationized samples were immersed in the dyeing solution and the dyeing was performed using the exhaustion method over 60 min under different conditions with the previously used exhaustion equipment. After dyeing, the samples were rinsed with tap water and dried at room temperature without being exposed to light. In this research, dyeing variables including temperature, pH, liquid ratio, and temperature rise gradient were optimised through experimental tests. The optimization of the dyeing variables was carried out sequentially in the following order: first the dyeing temperature, followed by pH, then liquid ratio, and finally the temperature rise gradient. Each parameter was individually optimised while keeping the others constant. The optimum temperature for dyeing was determined by testing different bath temperatures ranging from 40 to 70 °C. The optimum pH was found by testing different pH levels from 3 to 11. The optimum material-to-liquid ratio (MLR) was decided by testing different ratios from 1:10 to 1:50. The optimum temperature rise gradient was found by experimenting with different gradients from 1 to 4 °C/min. The optimal conditions were chosen based on bath exhaustion, and the colour strength and intensity of the dyed fabrics. In addition to the process conditions, different amounts of algae extracts were studied (2–5% wof). For clarity, samples were coded according to both the pre-mordanting treatment and the dyeing parameters in the following order: dyeing temperature, pH, liquor ratio, temperature rise gradient, and algae extract concentration.

2.2.4. Exhausting Curves

In addition to the dyeing evaluation, exhaustion curves were also performed and analysed to better understand the kinetics of dyeing and functionalization. For this purpose, calibration curves of each algae extract were first determined by preparing a series of solutions with different concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0 g/L) and measuring their peak absorbance. Dyeing experiments were then carried out under the optimal conditions defined in this study. During the dyeing process, 1 mL aliquots were collected at regular intervals between 0 and 100 min to construct the exhaustion curves. All experiments were conducted using the beaker dyeing machine (Tingiomat, Werner Mathis AG, Oberhasli, Switzerland).

The absorbance values were determined between 190–1100 nm (resolution of 1 nm) using the Shimadzu UV-1800 equipment (Shimadzu Corporation, Kyoto, Japan) and high precision quartz cuvettes of type 100-QS (Hellma Analyticals, Hellma GmbH & Co. KG, Müllheim, Germany) for each one of the extracts’ collected aliquots. The exhaust percentage using the following equation:

$$\mathrm{E}\mathrm{x}\mathrm{h}\mathrm{a}\mathrm{u}\mathrm{s}\mathrm{t}\left(\%\right)={\displaystyle \frac{{\mathrm{C}}_0-{\mathrm{C}}_{\mathrm{t}}}{{\mathrm{C}}_0}}\times 100$$

(2)

2.2.5. Fabrics Structural Characterization

Cotton fabrics before and after the mordanting process alongside the cationized knit and the functionalized fabrics were characterized. Fabrics’ morphology was assessed through optical microscopy using a Leica DM500 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Flexacam_15C digital camera (Leica Microsystems, Wetzlar, Germany). Fabrics were also analysed through UV–Vis spectroscopy using the Shimadzu UV–Vis-2600 equipment (Shimadzu Corporation, Kyoto, Japan) with the solids measurement system through all the spectrum range (220–1900 nm). In their turn, the fabrics’ chemical structures were verified through FTIR spectroscopy using the Alpha II spectrophotometer (Bruker Corporation, Billerica, MA, USA). For each sample, a total of 200 scans were performed at a spectral resolution of 1 cm⁻¹ over the wavenumber range of 4000–400 cm⁻¹.

2.2.6. Colour Assessment

The reflectance spectra of the mordanted and dyed samples were measured using a spectrophotometer (DataColor, Lucerne, Switzerland) in the visible light range of 400 to 700 with 10 nm steps. The colour strength (K/S) of the dyed samples was obtained performing three measurements at different positions on the knitted fabrics according to Kubelka–Munk [25]:

$${\displaystyle \frac{K}{S}}={\displaystyle \frac{{(1-R)}^2}{2R}}$$

(3)

where R is the reflectance value at each wavelength, K the absorbance coefficient, and S is the scattering coefficient. Also, CIE L*a*b* coordinates of the samples were measured according to ISO/CIE 11664-4 [26], where “L*” represents the darkness–lightness (0–100), “a*” represents the redness (positive value) and greenness (negative value), and “b*” represents the yellowness (positive value) and blueness (negative value). Total colour difference value ($∆E$) was calculated using the following equation, as reported by the same standard:

$$∆E={[{\left({∆L}^{\ast }\right)}^2+{\left({∆a}^{\ast }\right)}^2+{\left({∆b}^{\ast }\right)}^2]}^{{\displaystyle \frac{1}{2}}}$$

(4)

2.2.7. Colour Fastness

In this study, the colour fastness of the dyed samples was assessed using three different approaches: resistance to water (EN ISO 105-E01 [27]), domestic laundering (EN ISO 105-C06 [28]), and perspiration (EN ISO 105-E04 [29]). In addition, light fastness was evaluated using a QUV accelerated ageing chamber under continuous UV radiation for 4 h at controlled temperatures of 45 and 60 °C, with an irradiance of 0.89 W/(m²/nm) (adapted from ASTM G154, cycle 1 [30]). Colour changes were evaluated by measuring CIE L*a*b* coordinates and by comparison against the standard grey scale. To ensure consistency and relevance, fastness testing was conducted only on the optimised functionalized fabrics.

2.2.8. Antioxidant Properties

The AA of the optimised functionalized knitted samples was evaluated using the ABTS and DPPH radical cation decolourisation assay. In addition to the untreated functionalized samples, antioxidant activity (ABTS) was also assessed after one standardized washing cycle in order to evaluate the retention of functionality after laundering. Both radical solutions were prepared as previously reported. Functionalized samples were prepared by cutting 0.125 g of each fabric into small pieces. Briefly, 5000 μL samples of ABTS or DPPH working solution were added to each fabric sample and incubated in the dark for 30 min (ABTS) and 120 min (DPPH) under the same conditions. Absorbance was measured at 734 nm (ABTS) and 515 nm (DPPH) using the Shimadzu UV-1800 equipment (Shimadzu Corporation, Kyoto, Japan), and the antioxidant activity was expressed as the percentage inhibition of the radical solution relative to the control, as reported in Equation (1).

3. Results and Discussion

3.1. Extract Characterization

Both extracts were first characterized for their structural and thermal properties.

Both aqueous extracts exhibited distinct UV–Vis absorbance profiles, reflecting differences in their pigment composition and suggesting the presence of both protein-based (phycobiliproteins) and non-protein pigments (Figure 1), as reported by the literature [17,31,32]. A. platensis displayed strong absorbance bands around 350–450 nm and 600–700 nm, which can be attributed to chlorophyll a (with peaks near 400 and 660 nm) [22,33,34,35] and phycocyanin (around 620 nm) [22,36,37,38]. The region around 450–500 nm can also indicate the presence of carotenoids in smaller proportion [35,39,40]. In contrast, P. cruentum showed a sharp and intense peak centred around 550 nm, associated with phycoerythrin [22,41,42], a pigment typical of red microalgae. A secondary, smaller peak near 620 nm also suggested the presence of phycocyanin in this extract.

The FTIR spectra of A. platensis and P. cruentum revealed characteristic absorption bands typically found in natural compounds and algae biomasses, refs. [43,44,45,46,47,48] which can provide information on the functional groups present in these extracts (Figure 2). The overall similarity between the spectra reflects the structural resemblance of the predominant phycobiliproteins–phycocyanin in A. platensis and phycoerythrin in P. cruentum [32].

In line with previous FTIR studies on algae, the extracts exhibited prominent features associated with proteins, lipids, and carbohydrates [8,49,50,51,52,53]. The wide and noticeable band located at 3280 cm⁻¹ exhibited in both species revealed the presence of O–H and N–H stretching vibrations, indicating hydroxyl groups and protein-related amide structures [52,54]. The small peaks shown between 2960 and 2850 cm⁻¹ and at ~1450 cm⁻¹ can be associated with symmetric and asymmetric C–H stretching and bending in aliphatic chains (CH₂/CH₃ bending vibrations) characteristic of lipids components [53,55]. The intense bands between 1700 and 1500 cm⁻¹, corresponding to the amide I and amide II bands, are due to the C=O and C=N stretching and N–H bending, confirming protein structures [52,54] while the band at 1240 cm⁻¹ (amide III) can be associated with C–N stretching and N–H bending and may also reflect the asymmetric O=S=O stretching vibration from sulphate esters. A distinct band observed at approximately 1398 cm⁻¹ is attributable to C–O–O symmetric stretching vibration of carboxylate (–COO⁻) groups. The intense band at 1039 cm⁻¹ further confirms the presence of C–O, C–O–C, and C–C stretching possibly associated with carbohydrate-derived polysaccharides [50,51,56]. Finally, signals in the low-frequency fingerprint region (900–500 cm⁻¹) can be attributed to S–O and P–O bending vibrations as well as aromatic C–H groups from sulphated polysaccharides, commonly reported in both green [57,58,59] and red [60,61,62] algae species. Compared to P. cruentum, A. platensis appears to display more prominent amide bands, suggesting higher protein content, while both species showed similar carboxylate signals and slight differences in carbohydrate regions. Small peaks at 1120 and 923 cm⁻¹ were observed exclusively in A. platensis extract, likely reflecting C–O and C–O–C stretching of polysaccharides and possibly contributions from chlorophyll a and carotenoids consistent with UV–Vis data. Peaks around 2360 cm⁻¹ are characteristic of the CO₂ present in the surrounded air and so were excluded from this analysis. These results are consistent with previous literature on A. platensis [12,63,64,65,66,67] and P. cruentum [68,69] extracts.

The LC-MS analysis revealed that the extracts of A. platensis and P. cruentum present a diverse molecular composition, dominated by amino acids, organic acids, and aromatic metabolites (Supplementary Materials—Tables S1 and S2). LC-MS mainly allowed the characterization of the low-molecular-weight fraction of the extracts, which is known to play a key role in the biological and functional properties of algae [70].

In the case of A. platensis, the identification of several free amino acids indicates the presence of multiple polar and ionizable functional groups in the extract, such as amine and carboxyl groups. From a textile perspective, this composition is particularly relevant, as these groups can establish hydrogen bonds and, depending on the medium conditions, electrostatic interactions with cellulose, favouring the adsorption of the extract onto cotton fibres.

Similarly, the P. cruentum extract exhibited a chemically diverse composition, also dominated by polar metabolites. As observed for A. platensis, the presence of carboxyl and hydroxyl groups may promote intermolecular interactions, contributing to the retention of the extract on the fibre. The presence of compounds such as taurine, carnitine, and coumarin indicates a matrix rich in bioactive metabolites, which may exert antioxidant and protective effects. These compounds can add value to the natural dyeing process.

These extracts should be regarded as multifunctional chemical systems rather than simple natural colorants. Their molecular complexity enables multiple modes of interaction with cotton fibres, arising from the combined presence of chromophoric species and a wide range of polar and ionizable metabolites. This chemical richness not only contributes to colour expression but also introduces additional interaction pathways that can influence dye uptake, retention, and the overall behaviour of the fabric. As a result, the dyeing process becomes more than a mere coloration step, evolving into a chemically active system in which both aesthetic and functional attributes can emerge simultaneously. The thermal behaviour of A. platensis and P. cruentum extracts was evaluated through TGA and DSC analyses (Figure 3), enabling a detailed identification of their decomposition steps and comparison of thermal stability. Both extracts exhibited an initial mass loss below 100 °C of less than 10% (Figure 3a,b), corresponding to moisture evaporation and release of physically adsorbed water or residual solvents. This behaviour is typical of microalgae and other polysaccharide-rich biomaterials.

Following the dehydration step, both extracts displayed a major degradation region between 100 and 400 °C, consistent with the decomposition of thermolabile organic constituents such as pigments (phycobiliproteins, chlorophylls, carotenoids), polysaccharides, amino acids, and other protein fractions, in agreement with previously reported data for algae biomass and extracts [65,66,71,72,73,74,75,76]. Within this region, marked differences were observed between species. A. platensis presented two distinct DTG peaks at approximately 225 °C and 322 °C, resulting in a total mass loss of ~36%, indicating rapid degradation events characteristic of protein-rich cyanobacterial biomass. These peaks are compatible with the breakdown of phycocyanin and thermolabile peptide structures, aligning with literature reports of decomposition maxima in the 250–310 °C range for A. platensis. In contrast, P. cruentum exhibited a smoother and more gradual thermal degradation, with a slight peak at 229 °C and two greater DTG peaks at ~290 °C and ~314 °C, together corresponding to a weight loss of ~22%. This behaviour reflects the higher abundance of sulphated polysaccharides and the structural stability of phycoerythrin, whose denaturation typically occurs over a broader temperature interval. Red microalgae are indeed reported to undergo more progressive thermal decay due to their dense polysaccharide matrices. Above 400 °C, both extracts displayed residual decomposition associated with carbonisation and breakdown of recalcitrant aromatic structures, with final mass losses of ~59% for A. platensis and ~50% for P. cruentum. Overall, the thermal profile indicates that P. cruentum possesses greater thermal resistance, as reflected by its smoother DTG curve and lower mass-loss rate.

DSC analysis (Figure 3c,d) further supported the differences observed in TGA. In both extracts, a first endothermic peak near 100 °C was detected, assigned to moisture evaporation and disruption of weakly bound water molecules. Beyond 150 °C, both samples exhibited broad endothermic events associated with denaturation of proteins, cleavage of pigment–protein complexes, and decomposition of polysaccharides. A. platensis presented a more pronounced endothermic peak (onset at 88.6 °C, peak at 126.7 °C, ΔH = 216.94 J/g), while a second transition occurred at higher temperature (onset at 193.6 °C, peak at 220.1 °C, ΔH = 17.61 J/g). These sharper thermal events are in line with the DTG maxima and reflect the presence of more thermolabile protein fractions. Conversely, P. cruentum exhibited two endothermic transitions of lower intensity and broader shape, occurring at 101.8 °C (ΔH = 32.40 J/g, onset at 67.8 °C) and 213.4 °C (ΔH = 10.99 J/g, onset at 184.1 °C), which aligns with its more gradual mass-loss behaviour in TGA. These smoother thermal phenomena suggest that the structural organisation of sulphated polysaccharides and the stability of phycoerythrin confer enhanced resistance to abrupt thermal breakdown. Overall, DSC analysis corroborates TGA data, confirming that P. cruentum exhibits slightly higher thermal resistance, while A. platensis possesses more thermolabile constituents.

The AA of A. platensis and P. cruentum was evaluated using the ABTS and DPPH radical cation decolourisation assays (Figure 4). The results demonstrate a dose-dependent increase in ABTS radical scavenging activity for both microalgal extracts, with A. platensis consistently exhibiting higher inhibitory percentages across all tested concentrations when compared to P. cruentum. Conversely to the ABTS results, both A. platensis and P. cruentum extracts exhibited very low AA when tested with the DPPH assay. At the maximum concentration (2.5 mg/mL), A. platensis reached 92.06 ± 4.60% inhibition, while P. cruentum achieved a significantly lower but still notable value of 76.54 ± 2.27%. In contrast, the DPPH assay revealed very limited radical-scavenging activity for both species. Even at the highest concentration (2.5 mg/mL), inhibition values remained below 15%, reaching only 9.80 ± 0.51% for A. platensis and 14.83 ± 0.73% for P. cruentum. These results confirm that, although both extracts respond effectively in the ABTS assay, their antioxidant performance is markedly reduced when evaluated using the DPPH radical.

The stronger AA observed in the ABTS assay compared with DPPH can be explained by multiple factors:

• Solubility and radical accessibility—ABTS•⁺ is soluble in both aqueous and organic solvents, while DPPH• is lipophilic. This makes ABTS more suitable for detecting hydrophilic antioxidants. Also, ABTS•⁺ is relatively small and more accessible to a wide range of antioxidants, while DPPH• has a bulky structure that limits its reactivity with large or polar compounds [77].
• Reaction mechanism—Both ABTS•⁺ and DPPH• can react via electron transfer (ET) and hydrogen atom transfer (HAT); however, the kinetics differ. ET generally occurs faster than HAT in both assays, but reactions with DPPH• are slower overall, and HAT in particular proceeds very slowly. In contrast, ABTS•⁺ reaches equilibrium more rapidly, with both ET and HAT contributing comparably. This versatility allows ABTS assay to detect a broader range of antioxidant compounds and often results in higher measured activity values than DPPH [78,79].
• Method sensibility—ABTS is generally considered more sensitive to both low- and high-molecular-weight antioxidants, providing a more reliable representation of total antioxidant capacity in complex matrices.
• Extract methodology—The solvent system, extraction method, and conditions strongly influence the profile and concentration of antioxidant compounds present in the microalgae [80]. For instance, aqueous extractions favour hydrophilic constituents such as phycobiliproteins, phenolics, and polysaccharides, contributing to higher ABTS response. Conversely, DPPH activity tends to increase when organic solvents extract a greater proportion of lipophilic antioxidants such as carotenoids and certain fatty acids. The method of extraction (maceration, Soxhlet, reflux, supercritical CO₂, and ultrasound assisted) also influences the AA, as it can extract different amounts of antioxidant compounds [81].
• Complexity of algae extracts—Algae extracts contain mixtures of hydrophilic and lipophilic compounds. While ABTS•⁺ interacts with both, DPPH• mainly responds to lipophilic antioxidants, underestimating the overall antioxidant potential [77,82]. Considering that the extracts explored in this study were obtained through aqueous extraction, which favours the recovery of hydrophilic antioxidant compounds, and that the UV–Vis scan (Figure 1) confirmed the presence of hydrophilic pigments, the observed results are consistent with what is reported in the literature, where ABTS is expected to yield higher antioxidant values than DPPH for water-based extracts.

These results suggest that A. platentis is richer in different types of hydrophilic antioxidant compounds (chlorophylls, carotenoids, and phycobiliproteins), which explains its superior performance in the ABTS assay, while P. cruentum has phycoerythrin as the main constituent, which may lead to a lower antioxidant activity. In addition to hydrophilic compounds, P. cruentum may also contain higher lipophilic antioxidants than A. platensis, accounting for its slightly higher activity in the DPPH assay. This highlights the complementary nature of both assays and the importance of considering extract composition when evaluating antioxidant potential.

3.2. Fabric Functionalization

3.2.1. Pre-Treatment

After the mordanting process, the samples did not exhibit any significant change in colour, as confirmed by the CIE L*a*b* coordinates and whiteness degree. The whiteness loss was only 6.16% for the chitosan-treated samples and 1.50% for the alum-treated samples when compared with untreated cotton. Both remained noticeably higher in whiteness than the cationized sample, as shown in

Table 1. CIE L*a*b* coordinates and whiteness degree of cotton samples.

Sample	CIE L*a*b*	% White (Berger)	∆E
CO	92.74; −0.27; 3.54	65.91	-
CO_Al	92.64; −0.30; 3.72	64.92 (−1.50%)	0.84
CO_CS	91.14; −0.16; 4.12	61.85 (−6.16%)	0.21
CO_cat	93.83; −1.03; 5.65	59.59 (−9.59%)	2.49

Note: The colours shown in the table represent the visual approximation of the measured CIE L*a*b* coordinates, included to facilitate comparison of the resulting shades.

.

Microscopic analysis did not reveal significant morphological differences between unmordanted and mordanted fabrics. However, slight differences in surface appearance (Supplementary Materials—Figure S1) were observed in the commercial cationized samples, which exhibited a smoother surface with fewer loose fibres. This can be attributed to the unspecified cationic pre-treatment. The FTIR spectra of different samples are dominated by the characteristic absorption bands of cotton, namely at ≈3300 cm⁻¹ (O–H stretching), ≈2900 cm⁻¹ (C–H stretching), 1428 cm⁻¹ (C–H bending), 1315 cm⁻¹ (C–O–H bending) and strong C–O–C and C–O peaks in the 1200–900 cm⁻¹ region [83,84], as presented in Supplementary Materials—Figure S2a. No significant differences were observed between the FTIR spectra of untreated and functionalized fabrics. In particular, no new distinct absorption bands attributable to chitosan were clearly detectable. This may be explained by the relatively low add-on levels and by the fact that the functionalization is predominantly confined to the fibre surface. Additionally, characteristic chitosan bands may overlap with the strong cellulose absorption bands of cotton, limiting their detection by FTIR. Therefore, the overall spectra remain largely dominated by the characteristic cellulose framework. From UV–Vis absorbance scan (Supplementary Materials—Figure S2b) analysis, it is possible to infer that all samples display a gradual decrease in absorbance with increasing wavelength, a typical behaviour of cellulosic substrates in the UV region [85,86]. Although slight variations in absorbance were observed between untreated and modified fabrics, statistical analysis confirmed that these differences were not significant, indicating that the surface treatments did not substantially alter the overall optical response of the cotton substrates.

3.2.2. Dyeing

Table 2. K/S and CIE L*a*b* coordinates of the functionalized samples.

Sample Code	A. platensis		P. cruentum
	CIE L*a*b*	K/S Max	CIE L*a*b*	K/S Max
CO_40_7_20_1_2	90.97; −0.53; 5.16	0.0270	91.72; 1.06; 3.44	0.0300
CO_50_7_20_1_2	92.33; −0.76; 5.63	0.0189	92.42; 0.32; 3.97	0.0238
CO_60_7_20_1_2	92.02; −0.46; 5.52	0.0195	92.57; 0.52; 4.00	0.0233
CO_70_7_20_1_2	91.00; −0.42; 5.68	0.0257	92.28; 0.25; 4.06	0.0242
CO_40_3_20_1_2	88.13; −2.82; 6.56	0.0965	89.88; 4.20; 0.52	0.0501
CO_40_5_20_1_2	92.11; −0.46; 5.40	0.0186	83.02; 0.49; 3.69	0.205
CO_40_9_20_1_2	92.20; −0.68; 5.51	0.0198	92.78; 0.38; 4.26	0.220
CO_40_11_20_1_2	92.99; −0.46; 5.69	0.0160	93.05; 0.37; 4.64	0.0206
CO_40_7_10_1_2	91.81; −0.60; 6.71	0.0239	92.99; 0.09; 4.62	0.0206
CO_40_7_30_1_2	92.53; −0.72; 5.04	0.0170	93.57: −0.03; 4.25	0.0169
CO_40_7_40_1_2	92.48; −0.52; 6.83	0.0159	93.04; −0.39; 6.28	0.0197
CO_40_7_50_1_2	92.53; −0.61; 6.73	0.0156	93.14; −0.27; 5.73	0.0190
CO_40_7_20_2_2	92.24; −0.54; 6.83	0.0128	92.97; 0.18; 4.44	0.0206
CO_40_7_20_3_2	92.41; −0.65; 6.33	0.0174	92.81; 0.59; 4.30	0.0221
CO_40_7_20_4_2	92.94; −0.18; 5.21	0.0127	92.81; 0.65; 4.48	0.0223
CO_40_7_20_1_3	92.06; −0.67; 6.32	0.0210	92.34; 0.80; 4.36	0.0258
CO_40_7_20_1_4	91.60; −0.47; 6.71	0.0231	92.11; 1.2; 4.12	0.0281
CO_40_7_20_1_5	91.24; −0.54; 6.83	0.0259	92.41; 1.04; 4.31	0.0259
CO_Al_40_7_20_1_2	90.86; −0.47; 4.58	0.0226	89.50; 5.16; 1.44	0.0570
CO_Al_50_7_20_1_2	90.66; −0.56; 4.77	0.0238	90.40; 3.64; 2.92	0.0451
CO_Al_60_7_20_1_2	90.26; −0.18; 5.10	0.0255	86.40; 7.16; 0.37	0.0981
CO_Al_70_7_20_1_2	89.75; −0.13; 5.14	0.0285	87.64; 5.39; 1.12	0.0768
CO_Al_40_3_20_1_2	87.05; −3.41; 6.69	0.0965 *	91.28; 1.90; 2.41	0.0326 *
CO_Al_40_5_20_1_2	91.74; −0.78; 5.84	0.0227 *	86.01; 9.03; −1.92	0.1165 *
CO_Al_40_9_20_1_2	91.50; −0.60; 5.88	0.0224	91.61; 2.44; 3.47	0.0329
CO_Al_40_11_20_1_2	92.78; −0.26; 5.80	0.0142	92.04; 1.73; 4.27	0.0298
CO_Al_40_7_10_1_2	90.64; 0.82; −5.32	0.0236 *	89.63; 5.33; 1.70	0.0577 *
CO_Al_40_7_30_1_2	91.39; −0.72; 5.04	0.0212	90.47; 3.80; 2.01	0.0447
CO_Al_40_7_40_1_2	91.61; −0.36; 5.14	0.0189	90.07; 4.46; 3.28	0.0503
CO_Al_40_7_50_1_2	91.81; −0.28; 5.14	0.0178	90.09; 4.34; 3.04	0.0497
CO_Al_40_7_20_2_2	92.16; −0.34; 5.34	0.0159	89.69; 5.13; 1.82	0.0564
CO_Al_40_7_20_3_2	91.27; −0.38; 4.92	0.0211 *	90.23; 4.63; 2.50	0.0494 *
CO_Al_40_7_20_4_2	90.92; −0.64; 4.95	0.0224 *	89.91; 4.81; 1.83	0.0531 *
CO_Al_40_7_20_1_3	90.86; −0.83; 5.26	0.0241 *	89.53; 4.85; 2.16	0.0566 *
CO_Al_40_7_20_1_4	90.32; −0.92; 5.48	0.0277 *	89.11; 5.87; 2.22	0.0650 *
CO_Al_40_7_20_1_5	89.76; −0.98; 5.91	0.0325 *	90.51; 3.52; 3.23	0.0444 *
CO_CS_40_7_20_1_2	89.99; −0.98; 6.78	0.0390	89.29; 4.11; 2.98	0.0568
CO_CS_50_7_20_1_2	90.22; −1.00; 6.53	0.0376	89.04; 3.88; 3.03	0.0588
CO_CS_60_7_20_1_2	88.88; −0.87; 5.93	0.0461	87.08; 5.63; 2.40	0.0867
CO_CS_70_7_20_1_2	88.58; −0.42; 6.66	0.0528	86.08; 4.93; 2.14	0.0967
CO_CS_40_3_20_1_2	85.97; −3.19; 7.09	0.1109 *	85.28; 7.53; −1.23	0.1164 *
CO_CS_40_5_20_1_2	88.94; −1.59; 6.49	0.0572 *	80.59; 12.98; −3.77	0.2414 *
CO_CS_40_9_20_1_2	91.00; −0.82; 6.65	0.0328	90.42; 3.03; 3.98	0.4450
CO_CS_40_11_20_1_2	92.48; −0.34; 6.46	0.0158	91.72; 0.88; 5.02	0.0306
CO_CS_40_7_10_1_2	89.50; −1.43; 7.61	0.0481 *	81.73; 11.71; 1.27	0.0601 *
CO_CS_40_7_30_1_2	90.22; −1.42; 7.23	0.0437	81.62; 12.36; 0.88	0.0478
CO_CS_40_7_40_1_2	80.83; −0.75; 6.65	0.0289	82.41; 11.09; 3.69	0.0464
CO_CS_40_7_50_1_2	91.14; −0.78; 7.10	0.0263	82.24; 11.78; 2.99	0.0505
CO_CS_40_7_20_2_2	91.49; −0.78; 6.63	0.0234	89.37; 4.59; 3.01	0.0584
CO_CS_40_7_20_3_2	90.34; −1.18; 7.29	0.0400 *	89.07; 4.37; 2.69	0.0605 *
CO_CS_40_7_20_4_2	90.54; −1.30; 6.68	0.0352 *	89.06; 4.35; 2.79	0.0602 *
CO_CS_40_7_20_1_3	90.07; −1.15; 7.08	0.0404 *	88.86; 5.01; 2.92	0.0645 *
CO_CS_40_7_20_1_4	89.61; −0.93; 7.41	0.0434 *	88.89; 4.68; 3.31	0.0636 *
CO_CS_40_7_20_1_5	89.04; −1.21, 7.86	0.0515 *	88.57; 5.18; 3.30	0.0691 *
CO_cat_40_7_20_1_2	82.57; −3.98; 6.29	0.1524	81.79; 12.54; 1.02	0.2175
CO_cat_50_7_20_1_2	81.34; −3.75; 7.38	0.2001	80.41; 12.11; 1.89	0.2576
CO_cat_60_7_20_1_2	81.15; −2.90; 9.24	0.2357	81.72; 9.78; 3.98	0.2237
CO_cat_70_7_20_1_2	81.88; −2.35; 10.00	0.2155	81.97; 7.00; 4.59	0.1967
CO_cat_40_3_20_1_2	83.75; −3.78; 8.91	0.1564 *	82.03; 12.05; −1.65	0.1995 *
CO_cat_40_5_20_1_2	79.19; −5.43; 7.68	0.2757 *	79.57; 14.79; −1.54	0.2816 *
CO_cat_40_9_20_1_2	83.86; −2.86; 6.81	0.1273	91.39; 12.15; 1.89	0.2304
CO_cat_40_11_20_1_2	89.28; −0.94; 8.01	0.0506	85.35; 7.98; 4.90	0.1461
CO_cat_40_7_10_1_2	82.74; −3.57; 8.39	0.1691 *	81.73; 11.71; 1.27	0.2120 *
CO_cat_40_7_30_1_2	82.44; −4.30; 5.97	0.1450	81.62; 12.36; 0.88	0.2198
CO_cat_40_7_40_1_2	83.04; −3.76; 5.86	0.1332	82.41; 11.09; 3.69	0.2098
CO_cat_40_7_50_1_2	84.13; −3.67; 5.53	0.1089	82.24; 11.78; 2.99	0.2143
CO_cat_40_7_20_2_2	83.23; −4.09; 7.37	0.1450	81.77; 10.76; 1.66	0.2059
CO_cat_40_7_20_3_2	83.22; −3.37; 6.22	0.1357 *	78.82; 16.60; 0.32	0.3222 *
CO_cat_40_7_20_4_2	82.69; −4.20; 5.77	0.1496 *	83.27; 11.43; 1.93	0.1832 *
CO_cat_40_7_20_1_3	82.94; −3.85; 5.66	0.1289 *	81.31; 12.43; 1.55	0.2307 *
CO_cat_40_7_20_1_4	84.01; −3.12; 6.79	0.1124 *	81.64; 11.57; 2.53	0.2186 *
CO_cat_40_7_20_1_5	84.02; −2.89; 7.92	0.1141 *	82.09; 10.95; 3.15	0.2055 *

Note: The colours shown in the table represent the visual approximation of the measured CIE L*a*b* coordinates, included to facilitate comparison of the resulting shades; *—functionalized fabrics with visible staining and dye unevenness; Bold: the selected condition for this paper study.

presents the K/S values and the CIE L*a*b* colour coordinates of all functionalized fabric samples.

Both algae extracts demonstrated comparable interactions with untreated cotton, mordanted cotton, and cationized cotton substrates under all tested conditions. No visible coloration was observed on the untreated cotton samples, indicating minimal affinity of the dyes for the unmodified fibres. Cotton pre-treated with alum and chitosan exhibited light coloration suggesting some improvement in dye fixation. Among all treatments, cationized cotton fabrics showed the highest colour strength and resulted in more vivid and visually appealing colours for both algae extracts. This enhanced dye uptake can be attributed to the introduction of cationic sites on the fibre surface—although not detected in FTIR analysis—facilitating electrostatic interactions with the anionic or polar functional groups present in the algae dyes. While the mordanting process with alum and chitosan also introduces cationic sites on the cotton fibre surface, these sites were not sufficient to yield visually interesting or vibrant colours, as observed in the cationized fabric. Although the surface charge of the algae extracts was not experimentally measured in this work, literature reports indicate that Arthrospira platensis (also referred to as Spirulina platensis) typically exhibits a negative zeta potential at neutral pH [87,88]. Considering that the dyeing process was performed under near-neutral conditions, the enhanced colour strength observed for cationized cotton is therefore consistent with electrostatic attraction between negatively charged extract components and positively charged sites introduced on the fibre surface. While zeta potential data were not available for Porphyridium cruentum, the comparable dyeing behaviour observed for both algal systems under identical conditions suggests that similar electrostatic contributions may be involved. Even though direct surface charge measurements would provide further confirmation, the dyeing behaviour observed strongly supports the relevance of electrostatic contributions in the interaction mechanism.

Given these observations, the subsequent subsections focus exclusively on the cationized cotton samples, enabling a detailed evaluation of the influence of dyeing parameters such as temperature, pH, liquor ratio, temperature rise gradient, and extract concentration. Also, further experiments were performed only using cationized cotton fabric.

1.

Effect of Temperature

Higher temperatures led to increased colour strength; however, they also promoted degradation of the extracts, as indicated by a shift in hue toward browner tones, likely due to thermal breakdown of sensitive pigment compounds, even if such changes were not fully perceptible in the previous thermal analyses. In contrast, dyeing at 40 °C resulted in more vivid and brilliant colours, suggesting a balance between dye–fibre interaction efficiency and pigment stability. This behaviour was expected, since phycobiliproteins exhibit limited thermal stability. In fact, Hsieh-Lo et al. identified 45 °C as the maximum temperature at which these compounds remain stable [89]. To date, no studies have reported cotton dyeing specifically using A. platensis or P. cruentum extracts. Nevertheless, some works on cotton dyeing with isolated phycocyanin or phycoerythrin from other algal sources describe dyeing temperatures ≤ 65 °C, although without assessing the effect of temperature on the colour properties [9,49,90]. Additionally, a patent on phycocyanin-based dyeing recommends operating between 20 and 40 °C to avoid pigment decomposition, which further corroborates the results obtained in this study [91]. Therefore, 40 °C was selected as the optimum temperature for exhaust dyeing with these algae extracts.

2.

Effect of pH

Natural dyeing often requires controlled pH conditions since many natural pigments are sensitive to changes in acidity or alkalinity; neutral pH or slightly acidic/alkaline baths are frequently reported as optimal to preserve pigment stability and achieve consistent dye uptake [6]. The stability of phycobiliprotein pigments from algal extract, such as phycocyanin and phycoerythrin, has been shown to depend strongly on pH: neutral to slightly acidic/basic conditions favour pigment stability and colour retention [92]. In this work, as expected, neutral pH conditions resulted in the most consistent and visually appealing dyeing outcomes.

Across the tested pH range (3 to 11), clear visual changes in pigment appearance were observed (Figure 5), with both extracts, A. platensis (Figure 5a) and P. cruentum (Figure 5b), showing progressive colour alteration from pH 3 to pH 11 (1–5). These changes illustrate the sensitivity of phycobiliproteins to pH and support the dyeing behaviour observed. At low pH values (pH 3–5), the resulting shades were duller and often accompanied by unevenness in colour distribution. This may be attributed to the partial degradation or altered solubility of pigment components under acidic conditions as suggested by the literature [92] and as seen in Figure 5 (1),(2). On the other hand, alkaline conditions (pH > 9) caused visible fading and browning of the dyed fabrics, indicating possible degradation of the algal pigments, particularly those sensitive to high pH levels, as observed in Figure 5a,b (5). Neutral conditions (pH ~ 7) provided the best balance, ensuring pigment stability and effective interaction with cationized fibres, leading to brighter and more uniform coloration. The previously identified works involving phycocyanin or phycoerythrin extracts did not explicitly control pH, but the reported methodologies suggest that dyeing was generally performed under near-neutral conditions [9,49,90]. Therefore, pH 7 was selected as the optimal condition for preserving pigment integrity and enhancing dye–fibre interaction. These findings also suggest that both algae extracts possess a negative surface charge under neutral pH. This assumption can be confirmed by the literature on these extracts showing that the isoelectric point of phycobiliproteins is close to 6 [22], which confirms that, in fact, under neutral pH, the extracts exhibit a negative charge.

3.

Effect of Liquor Ratio

In natural dyeing, the liquor ratio strongly influences dye–fibre interaction because it affects dye concentration, diffusion behaviour, and bath exhaustion kinetics. High MLR is commonly used for dyeing with natural extracts because it leads to lower staining and greater colour evenness [93,94]. Among the tested ratios (1:10 to 1:50), the MLR of 1:20 was identified as the optimal condition, providing the best balance between colour strength and bath efficiency. At 1:10, although the dye bath was more concentrated, the samples showed noticeable staining and uneven dyeing, likely due to the rapid and uncontrolled exhaustion of the dye, as reported by multiple works on both natural and synthetic dyeing [93,95,96]. Increasing the MLR to 1:30 produced results very similar to 1:20 in terms of colour quality, but required more dye solution, making it less economical. Further increasing the MLR to 1:40 and 1:50 led to weaker coloration, as the dye became too diluted to ensure effective fibre interaction [94,95,96]. Given that the previously reported articles used MLR ≥ 1:40 [9,49,90], the present results indicate that 1:20 provides the best compromise between colour strength, evenness, and bath efficiency, making it the most suitable MLR for exhaust dyeing with these extracts, offering a more efficient and resource-saving alternative for exhaust dyeing with these algae extracts.

4.

Effect of Temperature Gradient

Although the effect of temperature rise gradient has not been specifically studied in the scientific literature for algae pigments, references on natural dyeing emphasise that a gradual increase in temperature is preferred to ensure proper diffusion and avoid uneven dyeing [97]. At higher gradients (≥3 °C/min), rapid heating resulted in uneven dye distribution, with visible patchiness on the fabrics, suggesting insufficient time for the pigments to migrate and properly interact with fibre sites during the heating phase. The intermediate gradient of 2 °C/min produced more consistent coloration; however, the slowest gradient (1 °C/min) provided the best combination of uniformity and colour depth, yielding K/S values of 0.1524 and 0.2175 for A. platensis and P. cruentum, respectively. Therefore, 1 °C/min was selected as the preferred gradient for exhaust dyeing with these algal extracts.

5.

Effect of Algae Extract Percentage

Natural dyeing frequently relies on high extract concentrations to achieve intense colour and good fixation; however, excessive amounts can cause surface deposition, poor penetration, and uneven dyeing, particularly when the fibre cannot fully absorb the pigment [97]. In this work, concentrations above 2% wof resulted in staining and uneven dyeing, without a corresponding improvement in colour intensity. This suggests that excess pigment may exceed the fibre’s absorption capacity, leading to surface deposition rather than effective fixation. In contrast, 2% wof yielded the most uniform and visually consistent results, with clean and homogeneous appearance across all substrates. Additionally, this concentration enabled a relatively high bath exhaustion, indicating efficient dye uptake and minimal waste of the extract. Therefore, 2% wof was selected as the optimal extract concentration, ensuring balanced dyeing performance without inducing blotching or oversaturation.

Taking all these findings into account, the optimal dyeing conditions for the functionalization of cotton fabrics with algae extracts were established as follows: dyeing temperature of 40 °C, pH 7, liquor ratio of 1:20, temperature rise gradient of 1 °C/min, and extract concentration of 2% wof. These conditions provided the best balance between the colour strength and uniformity with the process efficiency, while minimising pigment degradation and ensuring reproducibility. Notably, these results were achieved at a lower temperature and with a more concentrated bath than those reported in the literature for similar algae extracts [9,49,90], where slightly higher temperatures and larger liquor ratios were used. This demonstrates that efficient and uniform dyeing can be obtained under milder and more resource-efficient conditions.

3.2.3. Dyeing and Functionalization Exhaustion Curves

Figure 6 presents the calibration curves of the algae extracts (Figure 6a) and the corresponding dye exhaustion profiles on cationized cotton fabrics (Figure 6b). As shown in Figure 6a, both A. platensis and P. cruentum displayed strong linear relationships between absorbance and concentration (R² > 0.99), confirming the reliability of the calibration method for quantifying dye uptake. The exhaustion curves in Figure 6b reveal a rapid initial increase in dye absorption during the first 40 min, which can be attributed to the high concentration gradient between the dye bath and the fibre, together with strong electrostatic interactions between the algal pigments and the cationized cotton substrate. This initial stage is followed by a gradual decrease in the exhaustion rate, leading to a plateau after approximately 80–100 min, indicating that equilibrium is reached as available binding sites become progressively saturated and diffusion limitations increase. Both extracts exhibited comparable exhaustion behaviour, although P. cruentum showed slightly higher exhaustion percentages than A. platensis throughout the dyeing process, which may be related to differences in pigment composition and dye–fibre affinity. Although complete equilibrium is attained at longer dyeing times, a substantial proportion of dye uptake occurs within the first 60 min, supporting the effectiveness of the selected processing time and confirming that the main conclusions drawn from the dyeing experiments remain valid.

3.2.4. Fabric Characterization

1.

Structural

Functionalized fabrics were examined by optical microscopy to assess the distribution of algal extracts within the fibre structure. Microscopy images revealed that the colouration was not limited to the fabric surface but also penetrated the interior of the yarns, indicating effective dye exhaustion from the bath and diffusion of the dye molecules into the fibre structure, as exhibited in Figure 7. This observation supports the effectiveness of the optimised dyeing conditions in promoting not only surface adsorption but also deeper penetration, which is essential for improved colour fastness and durability of the functionalization. Additionally, the functionalized fabrics were analysed using FTIR and UV–Vis spectroscopy and compared with the original cationized cotton fabric (CO_cat) (Supplementary Materials—Figure S3). Similar to the mordanted samples, no significant structural changes were detected in the FTIR spectra, suggesting that the functionalization process did not alter the fundamental chemical structure of the cotton fibres. In contrast, the UV–Vis spectra of the functionalized fabrics exhibited characteristic absorption bands consistent with those observed in the aqueous extract solutions (reported in Figure 1), confirming the presence and successful fixation of algal pigments on the fibre surface while maintaining their optical features after the dyeing process.

2.

Colour Fastness

Table 3. Colour fastness results of cationized cotton fabric with A. platensis and P. cruentum.

	CO_cat + A. platensis		CO_cat + P. cruentum
	∆E	Grey Scale	∆E	Grey Scale
Domestic Laundering	4.39	3	4.79	3/4
Water	0.84	5	2.60	4
Perspiration (acid)	1.73	4/5	2.93	3/4
Perspiration (alkaline)	2.14	4	2.57	4
UV (45 °C)	7.32	1	10.74	1
UV (60 °C)	8.06	1	11.18	1

Note: The colours shown in the table represent the visual approximation of the measured CIE L*a*b* coordinates, included to facilitate comparison of the resulting shades.

provides the colour fastness results of the functionalized cationized cotton fabrics under different testing conditions, namely washing, water, perspiration in both acidic and alkaline media, and UV radiation (under 45 and 60 °C). The results are expressed in terms of CIE L*a*b* colour differences (ΔE) and the corresponding grey scale ratings.

The colour fastness assessment of functionalized cationized cotton fabrics indicates that both A. platensis and P. cruentum retained good stability under different testing conditions. In general, no staining was observed on adjacent multifibre fabrics, confirming fixation of the natural colourants to the cationized cotton substrate. The CIE L*a*b* colour difference (ΔE) values suggest that domestic laundering induced the most pronounced changes among the wet treatments, while water exposure resulted in minimal colour alteration, particularly for A. platensis, with colour differences of only 0.84. Perspiration tests in both acidic and alkaline media produced moderate colour changes, with slightly lower performance for P. cruentum.

In contrast, exposure to UV radiation resulted in significantly higher ΔE values compared to the other testing conditions, indicating that light was the most critical factor affecting colour stability. A slightly greater colour change was observed at 60 °C compared to 45 °C under constant irradiance, suggesting that temperature contributes to the degradation process in combination with UV exposure. This behaviour is consistent with the known photosensitivity of phycobiliprotein-based pigments, which are susceptible to photodegradation and structural alteration under UV irradiation.

Overall, these results demonstrate that cationization is an effective strategy to improve dye–fibre interactions and improve wet fastness performance. However, light exposure remains a key limitation for these natural pigment systems, highlighting the need for further optimisation (e.g., use of mordants, crosslinkers, or finishing agents) to enhance both wash and light durability.

3.

Antioxidant Properties

The AA of the functionalized cotton fabrics was also assessed using both ABTS and DPPH assays (

Table 4. Antioxidant activity of functionalized cotton samples through ABTS and DPPH methods.

Sample	ABTS Inhibition (%)	DPPH Inhibition (%)
CO_cat	9.48 ± 0.50	1.74 ± 0.80
CO_cat + A_platensis	68.13 ± 3.60	18.11 ± 0.81
CO_cat + P. cruentum	60.76 ± 1.18	11.91 ± 1.58
CO_cat + A_platensis (after 1 washing cycle)	42.40 ± 1.85	-
CO_cat + P. cruentum (after 1 washing cycle)	32.65 ± 2.31	-

). The untreated cationized control showed only residual inhibition (<10% in ABTS and <2% in DPPH), confirming that the textile substrate itself did not contribute to radical scavenging. In contrast, fabrics functionalized with A. platensis and P. cruentum displayed clearly higher activity, with ABTS inhibition exceeding 60% for both extracts. The DPPH assay yielded considerably lower values, remaining below 20%, with A. platensis showing slightly stronger activity than P. cruentum. These results are consistent with the trends observed for the pure extracts, confirming the successful transfer of bioactive compounds to the cotton substrate and highlighting the dependence of measured activity on the radical system employed. In addition, the AA of the functionalized fabrics was evaluated after one standardized washing cycle to assess the retention of functionality after laundering. After one washing cycle, ABTS inhibition decreased to 42.40 ± 1.85% for A. platensis and 32.65 ± 2.31% for P. cruentum. Although a reduction in antioxidant activity was observed, the fabrics retained measurable radical scavenging capacity, indicating that a fraction of the bioactive compounds remained associated with the textile substrate. This decrease in AA is consistent with the colour changes observed after laundering in the fastness tests, suggesting that partial pigment removal contributes to the reduction in antioxidant performance. The comparatively higher retained activity for A. platensis correlates with its slightly better colour stability, supporting the relationship between dye fixation and functional durability.

4. Conclusions

This study demonstrated the potential of Arthropsira Platensis and Porphyridium Cruentum algae extracts as natural colourants and functional agents for cotton knit fabrics. Through systematic optimisation of dyeing parameters—including temperature, pH, liquor ratio, temperature rise gradient, and extract concentration—efficient dye exhaustion and uniform colouration were achieved under mild conditions (40 °C, pH 7, M:L 1:20, 1 °C/min, 2% wof). Among the tested pre-treatments, the cationized cotton substrate exhibited the highest dye affinity, resulting in brighter and more saturated shades, confirming the role of surface charge in promoting dye–fibre interactions.

Microscopic analysis revealed that the dye was distributed not only on the surface but also inside the fibres within the yarn, confirming deep penetration. This indicates effective bath exhaustion and uniform dyeing throughout the yarn structure, which is important for colourfastness and consistency. Spectroscopic analysis (FTIR and UV–Vis) corroborated the successful deposition of algae pigments without structural damage to the cellulose substrate. The dyed fabrics displayed acceptable fastness to washing, water, and perspiration, particularly under neutral pH conditions, although exposure to UV radiation resulted in more pronounced colour changes, highlighting light sensitivity as a key limitation of these natural pigment systems. Antioxidant evaluation revealed that both algae extracts retained their bioactivity after the dyeing process, with higher radical scavenging activity observed in the ABTS assay compared to DPPH, reflecting the predominance of hydrophilic antioxidant compounds in the extracts and confirming their effective transfer to the textile substrate. After one standardized washing cycle, a reduction in antioxidant activity was observed; however, measurable radical scavenging capacity was maintained, indicating partial retention of bioactive compounds and supporting the relationship between dye fixation and functional durability

Overall, these findings demonstrate that algae-derived extracts can serve as dual-purpose natural dyes and bio-functional agents for cotton textiles. The combination of natural coloration and antioxidant functionality highlights their potential for multifunctional textile applications.

Beyond their capacity to confer colour to textile materials, the antioxidant properties of A. platensis and P. cruentum extracts add functional relevance to potential applications. Antioxidant activity is particularly advantageous in biomedical textiles such as wound dressings, where oxidative stress contributes to delayed healing, cellular damage, and inflammation. By scavenging reactive oxygen species, antioxidant-enriched materials may help promote a more favourable healing environment, while improving the stability of embedded pigments, lipids, and other bioactive compounds prone to oxidation. Moreover, natural extracts with antioxidant potential generally align well with biocompatibility requirements, supporting safe skin contact and reducing the risk of irritation associated with conventional synthetic additives.

Future research should focus on improving the colour fastness, particularly to washing and light, of naturally dyed textiles, which remain the key limitations of bio-based colourants. These enhancements can be pursued through advanced pre-treatment, post-treatment strategies, or by incorporating natural or bio-based auxiliaries that strengthen dye–fibre interactions and enhance pigment stability. The use of green crosslinkers, natural mordants, chelating compounds, or encapsulation techniques could further mitigate colour fading and enhance the durability of natural dyed fabrics. Beyond colour stability, future studies should also evaluate the long-term performance activity after laundering and exposure, as well as extend these approaches to other natural fibres and blends. Collectively, these strategies can pave the way for durable, multifunctional, and environmentally responsible textile systems.