New Maya Blue-like Pigments Obtained in the Presence of Green Seaweed Extract

Abstract

Nanopigments consisting of organic/inorganic hybrid composites represent a convenient alternative for pigments, combining the qualities of organic dyes with those of inorganic materials. We obtained Maya blue pigments from methylene blue and modified bentonites, using Ulva lactuca aqueous extract. Structural modifications of bentonites during the synthesis of nanopigments were evidenced by the interplanar distance and the basal peak position in X-ray diffraction patterns. The presence of methylene blue in Maya blue nanopigments was also confirmed by UV-vis and FTIR spectra. The adsorption study revealed a higher adsorption capacity in the presence of Ulva lactuca extract compared to water, and two adsorption models, Langmuir and Freundlich, were applied for studying the adsorption of methylene blue onto bentonite. The stability and seed germination effect of the resulting nanopigments were assessed. Accordingly, it was found that the nanopigments obtained in Ulva lactuca extract had superior redox stability compared to those obtained in water or methylene blue solution. The ability of bentonite-based nanopigments to be used in seed treatment is another interesting feature that can be exploited.

1. Introduction

Even while pigments have a number of advantages over dyes, some of the most common pigments, especially those based on toxic metals, are often replaced by organic dyes or hybrid dyes, which are inorganic materials that incorporate dyes [1]. Maya blue, a well-known artificial light blue pigment manufactured by the Maya in the beginning of the first millennium AD, is actually an organic/inorganic hybrid composed of indigo dye and palygorskite clay. It was utilized in pottery and mural paintings, being notable for its remarkable durability against various environmental factors, including resistance to the attack of organic solvents, oxidant and reducing agents, acids, bases, and UV irradiation [2,3]. The Maya blue-like pigments were later obtained by researchers from various clays and dyes and by different methods in order to obtain resistant, cheap, colored composites with low toxicity [3].

Inspired by the ancient Maya blue pigment, the immobilization of organic dyes in inorganic hosts has the advantage of the stabilization of the organic guest, being a method of transforming the soluble dye into an insoluble organic–inorganic pigment [4]. The nature of the inorganic host can be varied, and a range of clays and other inorganic materials such as zeolites, gamma-alumina, layered double hydroxides, silica, aluminum hydroxide, magnesium hydroxide, zinc oxide, etc., have been sensitized with natural and synthetic dyes [4,5,6,7,8]. Most often, the hybrid clay–dye nanopigments have been obtained by hosting cationic dyes in the interlayer space [5]. The hybrid pigments combine the requested color characteristics of the dyes with the properties of the inorganic matrix, such as good stability and barrier properties. Such intercalated hybrids were obtained from rhodamine or methylene blue and montmorillonite [1,9,10,11]. The clay–dye nanopigments could improve the dispersion of pigments in the matrix and, consequently, the final coating’s characteristics [12,13]. They have numerous benefits, including photosensitivity and color strength, and are utilized in coating, plastics, and printing inks. Because it is often desired to obtain a colloidal dispersion of pigment particles for printing ink applications, the particle size should be in the nanometer range [5].

In the present study, we obtained Maya blue-like nanopigments starting from an organoclay, i.e., modified bentonite, as host, and methylene blue (MB), a cationic dye, as guest, in different experimental conditions. Bentonite is an inexpensive, environmentally benign, and effective adsorbent material with a microporous structure and a high specific surface area. Montmorillonite, a 2:1 layered silicate, with two silica tetrahedral sheets and an aluminum octahedral sheet forming a sandwich structure that swells on contact with water, makes up the majority of bentonite composition. The substitution of silicon with Al(III) in the tetrahedral layers (the outer layers) and substitution of Al(III) in the octahedral layer (the inner layer) with Mg(II) or Zn(II) in the bentonite three-sheet structure results in a net negative surface charge, which can be balanced by inorganic exchangeable cations on layer surfaces [14,15]. Due to its properties, bentonite was used for obtaining hybrid materials by the intercalation of organic dyes like methylene green, methylene blue, acridine orange, crystal violet, thionine, rhodamines B, etc. [5]. By replacing the inorganic exchangeable cations in bentonites with organic cations in organobentonites, the clay surface is transformed from organophobic to strongly organophilic, resulting in an increase in adsorption capacity. Therefore, compared to natural bentonite, organobentonites are thought to be better adsorbents for organic molecules [14].

We used an organic derivative of a bentonite clay (i.e., dimethyldioctadecylammonium bentonite, Bentone 34), consisting in bentonite modified with dimethyldioctadecylammonium cations, as support for a cationic organic dye, MB. The commercial product Bentone 34 is used for many purposes, including paints, printing inks, fillers, waxes, etc. [16].

Methylene blue ([7-(dimethylamino)phenothiazin-3-ylidene]-dimethylazanium chloride, C₁₆H₁₈N₃ClS, MB) is an aromatic heterocyclic basic dye, namely a polymethine dye. MB has a characteristic deep blue color in the oxidized state, being colorless in the reduced form (leucoMB, LMB) [17]. In addition to its use as a dye, the most widely used dye in the textile sector, MB has many medical applications as a therapeutic agent in anemia, urinary tract infections, malaria, thyroid surgery, cancer treatment, plaque psoriasis, methemoglobinemia, antidepressants, cardioprotection, neuroprotection, for diagnostic procedures, as a photosensitizing agent to inactivate RNA viruses in plasma through photodynamic means, etc. [17,18]. The antimicrobial activity of MB is probably the most well-known pharmaceutical application [19]. In the case of MB-based composites, it is expected that, in addition to the characteristic blue color being better preserved in the inorganic matrix, MB will retain its properties that make it useful as a therapeutic agent, especially in topical applications. For example, the colloids based on MB and montmorillonite showed higher antibacterial activity than the pure MB solution [20].

We obtained, under mild conditions, Maya blue-like pigments based on bentonite and MB, which were stable to light and reducing agents, using a novel combination of inexpensive materials and reagents by valorizing vegetal waste. To obtain nanopigments with a large amount of MB with improved properties, an organobentonite was used as an adsorbent for MB. The resulting materials were explored as fertilizers for seed germination, but they could also be used in other applications.

As well, we proposed the synthesis of bentonite–MB nanocomposites by the cation exchange process in the presence of biomolecules extracted from the waste of Ulva lactuca (sea lettuce, UL), a green macro alga from the Black Sea, which can be considered “a source of troubles and potential riches” in the marine environment [21]. A high quantity of this alga is collected on beaches every year and, in recent years, studies on the valorization of this waste have become more important. Seaweed contains high amounts of polysaccharides belonging to the phycocolloids class, which can be found in cell walls and intercellular spaces. The main polysaccharide from Ulva lactuca is ulvan, a sulfated polysaccharide [22,23]. Seaweed waste is a very convenient source of water-soluble polysaccharides as surfactants. Based on the dependence of pigments’ color on their dimension, the biomolecules extracted from Ulva lactuca were introduced into the synthesis medium as potential surfactants. But an interaction of ulvan, which is an anionic polysaccharide, with bentonite and MB must also be considered, not only the reduction of the particle size.

The obtained clay–MB composites were characterized by X-ray diffraction (XRD), X-ray fluorescence (XRF), UV-vis and FTIR spectroscopy. Properties like photosensitivity, redox stability, and effect on seed germination were estimated for MB–clay nanopigments. The results confirmed a higher redox stability in the nanopigments obtained from Ulva lactuca extract, thus enhancing the importance of experimental conditions for obtaining nanopigments.

2. Materials and Methods

2.1. Materials

Chemicals for the synthesis and characterization of nanopigments were purchased from Carlo Erba, Cluj-Napoca, Romania (Bentone 34, B34), LOBA Feinchemie, Bucharest, Romania (white gelatin), and Sigma Aldrich, Bucharest, Romania (methylene blue, MB; ascorbic acid, C₆H₈O₆), and used as received, without further purification. The green seaweed (Ulva lactuca) was collected from the Black Sea beach, Mangalia (43.8109° N, 28.5869° E), Romania, in the summer of 2023.

2.2. Synthesis of Bentonite-Based Nanopigments

We obtained nanopigments in the presence of Ulva lactuca extract as a source of phycocolloids, as well as in water for comparison. To investigate the influence of surfactant molecules in modified bentonite, a Bentone 34 (B34) sample was used in the synthesis after calcination at 700 °C for 2 h. The calcination product was a reddish-brown powder (calcined bentone, CB), with a mass loss of 40%.

Ulva lactuca extract (ULE) was obtained as we previously reported [24,25]. In brief, the green seaweed (Ulva lactuca), collected as waste from the beach, was washed with tap water and then with distilled water, dried in an oven at 70 °C until a constant mass was achieved, hand-shredded, and sieved through a 4 mm mesh sieve. The obtained powder was mixed with water in a ratio of 1 g/100 mL and magnetically stirred for 2 h at 60 °C. The mixture was filtered through a cotton pad and the filtrate was further used in the synthesis of nanopigments.

Modification of bentonites with ULE. For studying the interaction between the bentonites and phycocolloids from ULE, both B34 and CB modified with ULE were obtained. A total of 1 g of B34, as well as 0.6 g of CB, were triturated with 5 mL of ULE, and the suspension was further diluted gradually with 95 mL of ULE and then sonicated for 15 min in an ultrasonic bath (MRC D80H Ultrasonic Cleaner; frequency: 43 kHz; ultrasonic power: 80 W). The mixtures were stirred for 2 h, at 60 °C, on an orbital shaker incubator (B. Braun Biotech International) and then filtered off. The samples were denoted B-UL and CB-UL, respectively. The color of the bentonite samples remained unchanged after the treatment with ULE, i.e., light beige (B-UL) and reddish-brown (CB-UL).

Synthesis of nanopigments. The bentonite–MB nanopigments were obtained in systems B34–MB and CB–MB, both in water and ULE. Furthermore, the adsorption process was studied, keeping in mind the influence of several parameters, of which the most important are the nature of reaction medium and the synthesis route, but also the ratio between clay and dye, temperature, and contact time. We followed two synthesis routes, i.e., “one-pot” and “one by one”, using both ULE and water.

Synthesis route 1—“one-pot”. B34 (1 g), respective CB (0.6 g) were triturated with 5 mL of ULE and with water. The resulting suspension was diluted with 95 mL of ULE, as well as with deionized water, sonicated for 15 min in an ultrasonic bath, and 100 mL of a 0.32 g/L (10⁻³ M) solution of MB was added (32 mg MB/1 g B34). The mixture was stirred for 2 h, at 60 °C, on an orbital shaker incubator. The blue powders were filtered off and dried. The samples were denoted B-MB 1 and CB-MB 1 (in water), respective B-MB 2 and CB-MB 2 (in ULE).

Synthesis route 2—“one by one”. B34 (1 g), as well as CB (0.6 g), were triturated with 5 mL of ULE. The resulting suspensions were diluted with 95 mL of ULE, sonicated for 15 min in an ultrasonic bath and stirred for 2 h, at 60 °C, on an orbital shaker incubator. A total of 100 mL of 0.32 g/L (10⁻³ M) solution of MB was added, and the mixture was further stirred for 2 h, at 60 °C, on an orbital shaker incubator. The blue powders were filtered off and dried. The samples were denoted B-MB 3 and CB-MB 3 (in ULE).

2.3. Adsorption Study

The adsorption of MB onto dimethyldioctadecylammonium bentonite (B34 organoclay) was studied both in ULE and water. For this purpose, 1 g B34 was triturated with 5 mL of water, as well as with ULE, and the resulting suspension was diluted with 45 mL of water, as well as with ULE. A total of 50 mL of MB aqueous solution in water (0.64, 0.96, 1.12, 1.28, and 1.44 g/L), as well as in ULE (1.28, 1.44, 1.60, and 1.76 g/L), were added, and the resulting mixtures were magnetically stirred at room temperature for 8 h. Afterward, the mixtures were kept overnight and filtered. The MB concentration in the filtrates was determined by measuring the absorbance of the MB solution at a 664 nm wavelength based on the calibration curve (y = 0.1571x − 0.0857; R² = 0.9943).

2.4. Characterization of Powders

The powders were investigated by X-ray diffraction (XRD) performed on a Rigaku Miniflex 2 diffractometer with Ni filtered CuKα radiation, in the range of 2θ, 6–60°, with a scan rate of 2°/min and a step of 0.02°. The X-ray fluorescence analysis was achieved using an Olympus InnovX Delta scanner with an Au/Ta anode and a solid-state Silicon Drift Detector (SDD). The UV-visible diffuse reflectance spectra of the powders were recorded, in the range of 220–850 nm, on a Jasco V 550 spectrophotometer with an integrating sphere, using MgO as the reference. The UV-visible spectra of solutions were recorded in the range of 200–900 nm, on a Jasco V 550 spectrophotometer. The FTIR spectra were recorded in the range 4000–650 cm⁻¹ on an Agilent Cary 630 FTIR spectrometer, ZnSe ATR mode.

2.4.1. Stability Studies

Two samples of nanopigments, obtained in water as well as in ULE, i.e., B-MB 1 and B-MB 2, were selected for the stability study, and the results were compared with a solution which contained the same quantity of MB. A total of 13 mg of each nanopigment was ultrasonically dispersed for 30 min in 100 mL of water and then it was brought to a final suspension volume of 250 mL. A total of 0.1 mL of an ascorbic acid solution (5 mg/mL) was added to 1.9 mL of each sample, and the concentration of MB in solution/suspension was monitored at 664 nm at intervals of 15 min each [18]. The experiments were conducted in triplicate. The MB concentration in the filtrates was determined.

2.4.2. Photosensitivity Assessment

For B-MB 1 and B-MB 2 samples, a photosensitivity study was performed by exposing them to visible light (20 W, mercury-vapor lamp) for 360 min. The absorption solution was monitored at 30, 60, 120, 180, 240, 300, and 360 min of exposure and compared with the initial value. The measurements were conducted in triplicate [18].

2.4.3. Effect on Seed Germination

For testing the effect of nanocomposites on seed germination, we used tomato seeds (Solanum lycopersicum L., var. Lillagro, from Agrosel Romania). A total of 0.40 g of gelatin was dissolved in 200 mL of H₂O at 40 °C and 20 mg of powder (B34, CB, B-MB 1, and B-MB 2) were added, and the resulting suspensions were magnetically stirred until the powders were evenly dispersed. After the suspensions were cooled to room temperature, the seeds were added, and the magnetic stirring continued for another 20 min. Each of the 20 coated tomato seeds were placed in Petri dishes (90 mm diameter), and uncoated seeds were used as a control. Each experiment was conducted in triplicate. The seeds were humidified with tap water (10 mL each pot) and treated with 10 mL of 1% solution of “Tomato grow” (a solution of humic acids, fulvic acids, phosphorus, potassium and microelements necessary for growth, from SemPlus Romania). The plots were kept in a greenhouse at 22 °C. The seed germination was characterized by mean germination time, mean germination rate, the coefficient of variation of the germination time, and the synchrony of germination [26,27].

Mean germination time (MGT) was calculated as the following [26,27]:

$$MGT={\displaystyle \frac{\sum _{i=1}^k{n}_i\times t_i}{\sum _i^k{n}_i}}$$

(1)

Mean germination rate (MGR) [26,27]:

$$MGR={\displaystyle \frac{\sum _{i=1}^k{n}_i}{\sum _{i=1}^k{n}_i\times t_i}}$$

(2)

Coefficient variation of the germination time (CV_t) [26,27]:

$${CV}_t={\displaystyle \frac{S_t}{t}}\times 100$$

(3)

Synchrony of germination (Z) [26,27]:

$$Z={\displaystyle \frac{\sum _{i=1}^k{C}_{ni,2}}{C_{\sum _{i=1}^{k}ni,2}}}$$

(4)

$$C_{ni,2}={\displaystyle \frac{n_i\times (n_i-1)}{2}}$$

(5)

where t_i is the time of the germination (day i); n_i—number of seeds germinated in the day i; k—the end of germination; S_t—standard deviation of the germination time; t—mean germination time [26,27].

2.5. Adsorption Isotherm Models

Based on the types of forces that exist between the adsorbent and the adsorbate, two types of adsorption processes are distinguished, namely chemical and physical adsorption. The amount of material adsorbed as a function of concentration at a constant temperature is described and predicted by adsorption isotherms [28]. Among them, the Langmuir and Freundlich models were applied in the study for the obtaining of nanopigments.

The adsorption process takes place at identical and equivalent definite localized sites in the Langmuir isotherm, an empirical model that assumes a monolayer adsorption of the molecules [29]. The linear form of Langmuir isotherm is represented as follows [29,30]:

$${\displaystyle \frac{1}{Q_e}}={\displaystyle \frac{1}{Q_{max}}}+{\displaystyle \frac{1}{K_L·Q_{max}{·C}_e}}$$

(6)

where Q_e = adsorbed amount at equilibrium (mg/g); Q_max = maximum adsorbed amount (mg/g); C_e = adsorbate concentration at equilibrium (mg/L); and K_L = adsorption equilibrium constant (L/mg).

The reversible and non-ideal adsorption process is described by the Freundlich adsorption isotherm model, which is used for multilayer adsorption and is not limited to the monolayer concept [28,29,30]. In a linear form, the Freundlich isotherm equation can be represented as [31]:

$$ln{Q}_e=ln{K}_F+n_F·ln{C}_e$$

(7)

where Q_e = equilibrium adsorption quantity (mg/g); K_F = adsorption capacity (mg/g); and n_F = adsorption intensity [30].

3. Results and Discussion

As a cationic dye, it was expected that methylene blue was adsorbed on bentonites, most probably by intercalation in the interlayer space, during a cation exchange process, in which the interlayer cations of the clay material were exchanged with other cations or organic molecules positively charged from the aqueous solution [32]. However, the utilization of medium-sized organic compounds can integrate numerous large molecules through a process of incremental expansion. The intercalation of organic compounds does not always entail the displacement of cations [33]. Also, the modification of clays with amphiphilic molecules like quaternary ammonium salts, as in dimethyldioctadecylammonium bentonite (B34), proceeds just because of their amphiphilic character. Thus, surfactant molecules present in the inorganic matrix generate a hydrophobic environment due to their hydrocarbon chains, enabling the retention of various organic molecules (such as MB or biomolecules from ULE). Meanwhile, the positively charged hydrophilic part of surfactant molecules (cationic head) facilitates the adsorption of anions through electrostatic interactions [34]. Ulvan, a negatively charged polysaccharide [35], could not interact with bentonite by ion exchange, but can interact because of their electrical charges. The interaction of MB with organic molecules from ULE was also possible, based on its positive charge and/or organic skeleton.

3.1. Characterization of Nanopigments

3.1.1. X-Ray Diffraction

Using the X-ray diffraction technique, the structural and layer spacing alterations that occurred in B34 and CB because of the interaction with ULE and the MB adsorption were examined (https://next-gen.materialsproject.org/materials/ (last accessed on 15 January 2025)). The XRD patterns of uncolored clays are presented in Figure 1.

The patterns indicate that B34 is composed primarily of montmorillonite (https://rruff.info/Montmorillonite/R110052 (last accessed on 15 January 2025)) [36]. For both B34 and B-UL, a characteristic peak of montmorillonite was observed at 2θ = 7.10° (d₀₀₁ spacing of 12.441 Å) and 6.66° (d₀₀₁ spacing of 13.265 Å), respectively, and another well-defined and very intense peak was found at 19.77° (d₁₁₀ = 4.487 Å) and 19.68° (d₁₁₀ = 4.507 Å), respectively. Other peaks with lower intensity are also assigned to montmorillonite [36,37]. The peak at 27.52° in the B34 pattern, and at 26.52° for B-UL, can be assigned to quartz, which is the second component of bentonite (https://rruff.info/Quartz/R100134 (last accessed on 15 January 2025)). The calcination of B34 highlighted the peak, situated at 18.66°, in the XRD pattern of the CB sample, which shifted to 18.40° for CB-UL. This can also be attributed to montmorillonite. Additionally, there was a higher intensity for the peaks at 28.06° and 27.85°, which are less intense in XRD patterns of uncalcined bentonite. The low intensity peak at 10.50° in the XRD patterns of B34 and B-UL, which can be assigned to organic molecules, disappeared through the calcination of the organoclay.

The presence of organic compounds intercalating into layers of clay can be correlated with the interplanar distance, d, in XRD patterns [37,38,39]. Prior research has shown that, depending on their structure, the surfactant ions in organobentonites (such as dimethyldioctadecylammonium in B34) can be found both at the outer surface and in the interlayer area [34]. The shifting of basal peak to lower values of Bragg angle in the B-UL sample and the increase in the calculated interplanar distances are evidenced for the interaction between B34 and ULE. Although the most intense peak remains situated at about 19.7° in both XRD patterns, the relative intensity for all peaks is different in the XRD pattern of the B-UL sample in comparison with the pattern of B34. Furthermore, it was demonstrated that the surfactant cations might create deposition sites, where more surfactant ions would be bound by hydrophobic interactions [34]. Therefore, a hydrophobic interaction between the organic surfactant cations deposited onto the B34 surface and biomolecules from ULE is also possible. By calcining the B34 organoclay, the interplanar distance d₀₀₁ decreased from 12.441 Å (B34) to 9.550 Å (CB), which can be associated with the depletion of the organic and water molecules that have been intercalated. Further, the d₀₀₁ value increased to 9.794 Å after the modification of CB with ULE (CB-UL), probably because of the intercalation of biomolecules.

The XRD patterns of nanopigments based on B34 and CB are displayed in Figure 2.

The shape of XRD patterns for nanopigments based on B34, as well as for CB (Figure 2), is similar to that of the corresponding clay, proving the presence mainly of the montmorillonite phase. The weak and broad peak, which is found at 2θ = 10.50° in the case of B34, is negligible in the XRD patterns of blue clays, as well as in the case of B-UL. The organic cation from B34 was probably mostly removed by the further treatment of B34. Depending on the chloride ion location, the reported values for the MB molecule were 13.82 or 14.47 Å for the length and approximately 9.5 Å for its width [30]. The width of the MB molecule indicates that it can be accommodated in the structure of B34, relating to the d₀₀₁ spacing value. The shifting of peaks in the blue clay samples and the increase in the calculated interplanar distances can be correlated with such an interaction between B34 and MB. Another possibility is MB adsorption onto the clay surface. Previous studies demonstrated, by using SEM and EDS techniques [40], the presence of MB species on the clay surface; thus, the adsorption mechanism is explained by the electrostatic attraction between the positively charged dye molecules and the negatively charged silanol groups at the edges of clay surfaces [40,41]. The large amount of MB adsorbed onto bentonites, especially in the presence of ULE, constitutes an argument for the relevance of the second mechanism.

By comparing the B34-based nanopigments, the highest value of d₀₀₁ was determined for B-MB 2 (13.541 Å), probably because of several types of adsorbed molecules. The B-MB 2 nanopigment was obtained by the “one-pot” method; thus, the ULE and MB were added together, without giving the organic cations from B34 (i.e., dimethyldioctadecylammonium) the possibility to be desorbed beforehand. Consequently, the d₀₀₁ value is the largest of the interplanar distance values determined for the basal plane, for both uncolored and colored clays. A similar technique was followed for the synthesis of B-MB 1, and a lower value of d₀₀₁ compared to the untreated B34 (12.949 Å vs. 12.441 Å) was determined. However, in the absence of biomolecules from ULE, the value remains lower than for B-MB 2 (12.949 Å vs. 13.541 Å). The B-MB 3 nanopigment was obtained in two steps, the first step allowing the exchange between the intercalated cations from organoclay and biomolecules from ULE. Consequently, following the gradual interaction of organoclay with ULE/MB, the basal distance changed less compared to the untreated organoclay, and remained lower than the value for B-MB 2 (12.809 Å vs. 13.541 Å). A similar increase in the d₀₀₁ value after the adsorption of MB was evidenced for CB-based nanopigments also (for example, 10.183 Å for CB-MB 1 compared to 9.550 Å for CB or 9.794 Å for CB-UL). Moreover, in the simultaneous treatment of clay with both MB and ULE, the ability of MB to interact with ulvan, an anionic polysaccharide, should not be neglected, since the obtaining of the ionic pair of anionic surfactant–MB is an established method for quantitatively determining the presence of anionic surfactants in water [42]. Likewise, the insertion of organic molecules into the clay structure led to an increase in the crystallite size of the nanopigments.

3.1.2. X-Ray Fluorescence

The mineral composition of clays and nanopigments, obtained by XRF analysis (

Table 1. Chemical composition (% by weight) of clays and clay-based nanopigments.

Sample	Constituent Concentration (%)
	SiO2	Al2O3	Fe2O3	S	Other Oxides
B34	63.41 ± 0.26	24.21 ± 0.29	11.86 ± 0.09	0.15 ± 0.02	0.37
CB	63.42 ± 0.24	25.20 ± 0.27	11.06 ± 0.08	-	0.32
B-MB 1	61.35 ± 0.25	23.93 ± 0.28	11.80 ± 0.08	1.80 ± 0.03	1.12
B-MB 2	59.76 ± 0.25	24.06 ± 0.29	12.71 ± 0.09	2.53 ± 0.04	0.94
B-MB 3	60.16 ± 0.24	24.09 ± 0.28	12.28 ± 0.08	2.50 ± 0.03	0.97
CB-MB 1	61.82 ± 0.21	25.68 ± 0.24	10.41 ± 0.07	1.44 ± 0.02	0.65
CB-MB 2	61.38 ± 0.22	25.36 ± 0.25	11.26 ± 0.07	1.50 ± 0.03	0.50
CB-MB 3	60.48 ± 0.23	25.39 ± 0.27	12.09 ± 0.08	1.55 ± 0.03	0.49

), revealed that the main elements in clays are Si, Al, and Fe. The presence of iron was also confirmed by UV-vis spectroscopy through the characteristic transitions assigned to Fe(III) (Figure 3). The ratio of the main elements was not changed by calcination, but a slight variation can be observed after subsequent treatments, meaning a decrease in silicon along with an increase in Fe content. The sulfur content increased due to the adsorption of MB, both in the synthesis in water and in ULE. A higher quantity of sulfur in the B34-based nanopigments obtained in ULE can be explained by the presence of sulfate groups in ulvan. A similar, but smaller variation could also be observed for the CB-based nanopigments.

3.1.3. UV-Vis Spectroscopy

Figure 3 displays UV-vis spectra for the clay-based composites. The very strong band situated in the range of 250–253 nm in the spectra of uncolored clays (Figure 3a) is assigned to a charge transfer transition, i.e., oxo → Fe(III) (Oh), characteristic of montmorillonites. The bands and shoulders in the 300–530 nm range, more intense for calcined bentonite, are assigned to octahedral Fe(III) (Figure 3a) [43]. These absorption bands in the visible domain are responsible for the reddish-brown color of calcined bentonite, being characteristic of clays containing iron [44]. A very weak absorption band in the 650–700 nm range can be observed (Figure 3a), which is due to the adsorption of colored compounds from natural extracts, most probably chlorophyll a [45].

The bands assigned to MB can be found in the spectra of the nanopigments (Figure 3b). The visible spectra of the dyes adsorbed onto clays were obtained by the subtraction of the clay spectra from those of the nanopigments (Figure 3c).

The distinctive deep blue color of MB in the oxidized form is due to chromophore and auxochrome groups. An MB monomer is ascribed to the most intense band in the UV-vis spectrum of MB, which is found at about 664 nm; an MB dimer is assigned to the band or shoulder at about 612 nm. Assuming a monomer–dimer equilibrium, it was shown that the absorption spectra of aqueous MB solutions could be quantitatively analyzed [17,46]. The presence of a trimer was also observed in the MB solution [47].

All the B-MB and CB-MB composites have a deep blue color (Figure 3b), regardless of the method for obtaining them. The most intense band, resulting from the overlapping of two bands, is broad and shifted. Small differences in the position of the absorption maximum can be observed (Figure 3b). In the spectra of nanopigments based on CB, an additional band at approximately 760 nm was identified. The bands situated in the UV domain in the blue clays, i.e., at 280–290 nm, are assigned to the substituted benzene rings [17].

The shape of the adsorbed MB spectrum (Figure 3c) is quite different from that of MB dissolved in water (Figure 4b). The absorption band, situated in the visible domain at 550–700 nm for MB in aqueous solution, is broader (400–800 nm) in the spectra of adsorbed MB. The most intense band, at 662 nm in MB solution spectrum (Figure 4b), has a different shape in the adsorbed MB spectrum. The intensity of the band/shoulder, at about 612 nm, becomes higher, turning into a band which is partially overlapped with the other intense band and has approximately the same intensity (Figure 3c), the poor separation of bands being due to the high concentration of MB [46]. So, in the spectra of adsorbed MB, two intense bands with very similar intensity and partial overlapping, centered around 597 and 657 nm and assigned to (MB⁺)₂ and MB⁺, respectively, can be observed. The absorption maxima are more evident in the spectra of the blue clays obtained in water; the two bands almost turn into one in the spectra of the samples obtained in ULE. The band situated at about 760 nm in the spectra of MB adsorbed onto CB can be assigned to the protonated MB, i.e., MBH²⁺ [45]. An aggregation of MB molecules in higher polymers (n-mers) is also possible, as it has been reported for concentrated solutions [46,47]. The bands assigned to monopositive monomer species and the dimer are more intense compared to those assigned to the dipositive monomer [46]. A slight difference in blue hue can be observed between B– and CB–based nanopigments, probably because of the presence of protonated MB.

The resulting spectra of the supernatants after the removal of nanopigments (Figure 4a) are quite different, both qualitatively and quantitatively. Thus, the band intensity can be correlated with the adsorption capacity of bentonites under different conditions, but also with the presence of nanopigment particles in suspension. Based on the intensity of bands assigned to (MB⁺)₂, MB⁺, and MBH²⁺, a different ratio between MB species, and hence a different composition, has been observed for supernatants compared to the initial solution. For example, the low intensity shoulder situated at about 760 nm in the spectrum of CB-based nanopigment supernatants (Figure 4a) proves the presence of MBH²⁺, but this species cannot be identified in the spectrum of the MB solution (Figure 4b). An explanation can be the protonation of MB⁺ in solution in the presence of CB, or, more probably, the presence of nanopigments in the suspension, the highest quantity being estimated for CB-MB 3.

3.1.4. FTIR Spectroscopy

Figure 5 displays the FTIR spectra of uncolored clays and nanopigments.

The Si–O–Si bending vibration in the tetrahedral sheet is the cause of the very strong absorption band at 1002 cm⁻¹ that dominates the FTIR spectrum of B34 (Figure 5a) [36]. The Al–OH and silanol groups are responsible for the band seen at 913 cm⁻¹ in the FTIR spectrum of B34. A very weak vibration at 798 cm⁻¹ indicates the presence of quartz in B34, as was suggested by X-ray diffraction. The broad band at 3630 cm⁻¹ is assigned to hydroxyl groups, which are probably associated by hydrogen bonding. The band at 1636 cm⁻¹ in the B34 spectrum is responsible for adsorbed water molecules [36,48]. The bands at 883, 1468, 2848, and 2918 cm⁻¹ of B34 are assigned to the methylene groups [49]. The bands assigned to organic compounds are also observed in the B-UL spectrum (Figure 5a). The thermal modification of B34 has the effect of slightly shifting the most intense band assigned to the Si–O-Si bending vibration (Figure 5a), probably because of structure modification. The FTIR spectra of the uncolored clays (Figure 5a) are not relevant for the presence of natural surfactants from ULE [23], because similar functional groups are already present in the initial organoclay B34. It can be seen clearly how the modification of clay with ULE led to changes in the FTIR spectrum for CB. Thus, the CB FTIR spectrum shows that, by calcining B34, all the bands characteristic of organic compounds disappeared, leaving only a very weak band that can be attributed to water molecules at 3671 cm⁻¹ and the bands characteristic of the inorganic structure, namely the most intense band at 1010 cm⁻¹ (slightly shifted compared to B34), a medium-intensity band at 935, and weak bands at 798 and 1122 cm⁻¹ [50]. After the modification with ULE, the bands characteristics to organic molecules can be found again at 2922 and 2848 cm⁻¹ (C-H asymmetric and symmetric stretching). The spectra of colored clays (Figure 5b) also revealed the presence of organic compounds, but the intensity of absorption bands assigned to organic compounds is lower in comparison to the inorganic matrix. However, the bands characteristic to MB (Figure 5c), situated at 1584, 1483, and 1379 cm⁻¹, can be identified in the nanopigment spectra (Figure 5b) and have very low intensity. Furthermore, a very weak band, situated at 1245 cm⁻¹ (for CB) and at 1249 cm⁻¹ (for B34), in the spectra of the nanopigments obtained in ULE, can be assigned to S=O bond vibrations of ulvan [48]; however, MB also has a characteristic absorption band at 1241 cm⁻¹. The bands assigned to C-H and O-H bonds are also found in the FTIR spectra of nanopigments based on B34, at similar wavenumbers, indicating the presence of surfactants in the nanopigment composition.

Stability studies

The clay–MB composites with a nice blue color can be used as pigments in paints and coatings. In such applications, the stability of MB is an important issue, but MB is vulnerable to the redox process, which changes the oxidized blue form into leucomethylene blue, a reduced colorless form (Figure 6) [17].

To study the stability of adsorbed and/or released MB toward the reduction process, the reduction with ascorbic acid was performed and the color of the suspensions was monitored [18]. Two nanopigments, i.e., B-MB 1 and B-MB 2, were tested in this experiment in comparison with an MB solution, with all three samples containing the same quantity of MB (Figure 7). The first observation, before the redox process, concerns the color of the nanopigment suspension. As the UV-vis spectra revealed, the B-MB 1 sample released a higher quantity of MB in the solution than B-MB 2, although equivalent quantities of powders were used. This means there was an easier release of MB from the composite obtained in absence of ULE, and a stronger interaction between clay and MB for the composite obtained in ULE.

The concentration of MB solution decreased by around 89% after 15 min of exposure to a reducing agent (ascorbic acid) and then it was steady (Figure 7a), while the concentration of B-MB 1 supernatant solution decreased by 73.5% and the concentration of B-MB 2 decreased by only 57%, proving the highest stability to reduction, probably due to the presence of biomolecules from ULE. The photostability of B-MB suspensions was lower compared to that of MB solution (Figure 7b), but the B-MB 2 sample proved to be more stable than the B-MB 1 sample under light.

Effect on seed germination

In this study, we propose a new application for clay-based nanopigments, namely in the treatment of seeds. Numerous studies have been dedicated to the biological effects of nanomaterials, including clays and organic compounds, on plant germination and seedling growth, the nano-agriculture being able to provide a gainful impact to the crop [26]. In addition to the effect on seed germination, it is expected that these nanopigments containing MB will also act as disinfectants, thus protecting seeds in their natural environment (in the soil).

We tested the effect of nanopigments on tomato (Solanum lycopersicum L.) seed germination. We calculated germinability (G), mean germination time (MGT), the coefficient of variation of the germination time (CV_t), mean germination rate (MGR), the uncertainty of the germination process (U), and the synchrony of the germination process (Z) (Equations (1)–(5)). The results are presented in

Table 2. Germination measurements of Solanum lycopersicum L. seeds using bentonite-based materials.

Sample	G (%)	MGT (day)	CVt (%)	MGR (Day−1)	U (Bit)	Z
Control	90.00 ± 4.08	3.39 ± 0.31	51.77 ± 10.12	0.2971 ± 0.0312	2.52 ± 0.27	0.12 ± 0.01
B34	90.00 ± 4.08	2.67 ± 0.30	38.98 ± 7.25	0.3776 ± 0.0407	1.81 ± 0.21	0.21 ± 0.02
CB	90.00 ± 9.43	4.11 ± 0.37	24.39 ± 6.55	0.2478 ± 0.0199	1.50 ± 0.12	0.33 ± 0.03
B-MB 1	90.00 ± 7.07	3.21 ± 0.31	23.24 ± 6.44	0.3180 ± 0.0345	1.27 ± 0.15	0.41 ± 0.04
B-MB 2	90.00 ± 7.07	3.72 ± 0.35	21.64 ± 6.27	0.2786 ± 0.0256	1.47 ± 0.13	0.35 ± 0.03

.

The values of MGT and MGR can be correlated with seedling robustness. The coating of seeds with modified bentonites changed the MGT, with a higher decrease for B34 and a lower one for B-MB 1, but a higher increase for CB and a lower one for B-MB 2. By comparing the effect of B-MB 1 and B-MB 2, it can be seen that the B-MB 1 powder is more effective, resulting in MGT declining. The increased rate of seed germination may be explained by nanomaterials’ ability to pierce the seed coat and activate the embryo by causing the breakdown of enzymes associated with seed dormancy [27]. This was considered as faster seed germination may cause two main metabolic processes: activating respiration and quick ATP generation [51]. As in B34 and nanopigments, organic molecules’ presence seems important for germination rate. A measure of germination speed is the coefficient variation of mean germination time, which rises as more seeds germinate and falls as germination time increases [27]. The highest CV_t value was determined for B34, but all the values determined for the bentonite-based samples are lower than that of the control. The synchrony of germination is an indicator of the homogeneity of the germination process, with higher synchronous germinated seeds for the treatment with B-MB 1 and, to a lesser extent, B-MB 2. Compared to the control, a decrease in mean germination time, the coefficient of variation of the germination time, and the uncertainty of the germination process, as well as an increase in the mean germination rate and synchrony of the germination process, were observed for B-MB 1. For B-MB 2, was observed an increase in the mean germination time and synchrony of the germination process, while the coefficient of variation of the germination time, mean germination rate, and uncertainty of the germination process decreased.

This preliminary study, which showed the ability of bentonite and MB-based nanopigments to be used for seed germination, deserves to be continued in order to determine the optimal amount of bentonite-based materials and the conditions for seed treatment.

Adsorption study

The synthesis of nanopigments using the “one-pot” method was the starting point for a study on MB adsorption onto B34, both in aqueous solution and in ULE. Considering that the synthesis time was adequate to achieve adsorption–desorption equilibrium, the clay adsorption capacity at equilibrium (Q_e, mg/g) was calculated as follows [52,53,54]:

$$Q_e={(C}_i-C_e){\displaystyle \frac{V}{m}}$$

(8)

where C_i is the initial dye concentration (mg/L), C_e (mg/L) is the equilibrium concentration, V is the volume of the dye solution (L), and m is the mass of adsorbent (g).

The adsorption capacity of B34 was studied in aqueous solution, in comparison with ULE, at different MB concentrations. In aqueous solution, the initial quantity of MB (i.e., 32, 48, 56, 64, and 72 mg for 1 g of B34) was adsorbed almost completely onto B34, the calculated values of adsorption capacity, Q_e, being 31.99, 47.98, 55.90, 63.73, and 71.63 mg/g. A similar behavior was observed for ULE, the Q_e calculated values being 63.92, 71.92, 79.90, and 87.88 mg/g for initial ratios of 64, 72, 80, and 88 mg/1 g B34. By comparing the adsorption capacity of B34 in water and ULE for the same MB concentration, higher values were found in ULE than in water, i.e., 63.92 vs. 63.73 mg/g, and 71.92 vs. 71.63 mg/g, respectively.

Adsorption isotherms

The MB adsorption process was subjected to the Langmuir and Freundlich isotherms (Figure 8 and

Table 3. The Langmuir and Freundlich adsorption isotherm parameters.

Adsorption Conditions	Langmuir			Freundlich
	Qmax (mg/g)	KL	R2	nF	KF (mg/g)	R2
water	76.9231	1.0656	0.9916	0.3146	3.6561	0.9614
ULE	322.5806	0.1640	0.9104	0.7452	3.8534	0.9230

). The R² values demonstrated that both models are appropriate for the aqueous solution of MB, with the best fit being obtained for the Langmuir’s model (R² = 0.9916). In ULE, the processes become more complicated, with MB adsorption interfering with that of biomolecules from ULE, which can also interact with MB. Consequently, the values for R² are lower, and the best-fitting model is Freundlich’s model (R² = 0.9230). Therefore, MB adsorption in water is most likely monolayer adsorption onto the B34 surface, whereas MB adsorption in ULE is most likely multilayer adsorption on rough and multisite surfaces. The multilayer MB adsorption onto organoclay is consistent with the findings of other authors [34], namely the effect of surfactants to create deposition sites where more organic molecules can be bound by hydrophobic interactions. The effect is also favored by the presence of ulvan, which is also a surfactant, and can be an explanation for the higher adsorption capacity of organobentonite in ULE.

The Langmuir constant, K_L, which indicates the degree to which the adsorbate and surface interact [29], shows a weaker interaction between MB and the clay surface in ULE compared to H₂O. Instead, the calculated value of the maximum adsorption amount is much higher in ULE. Furthermore, the Langmuir model is less appropriate for adsorption in ULE.

The separation factor (R_L) is a dimensionless constant which is defined as [29]:

$$R_L={\displaystyle \frac{1}{1+K_L·C_0}}$$

(9)

where C₀ is the initial concentration of the adsorbate in (mg/L) and K_L, is the Langmuir constant. The calculated R_L values ranged from 2.92 × 10⁻³ to 1.30 × 10⁻³ (in water), and from 9.44 × 10⁻³ to 6.88 × 10⁻³ (in ULE), the adsorption being therefore favorable in both water and ULE, in the studied domain of concentration [31].

4. Conclusions

We succeeded in obtaining Maya blue-like pigments, in mild conditions, by using bentonite as the inorganic matrix and methylene blue in a phycocolloid. Thus, we obtained and characterized four new nanopigments, based on bentonites and methylene blue, either in water or in the presence of an Ulva lactuca extract. The nanopigments were obtained following two methods. The characterization of the nanopigments revealed that the synthesis route had only a small influence on the properties of the nanopigments, namely in the crystalline structure. The X-ray diffraction patterns, as well as UV-vis and FTIR spectra, confirmed the synthesis of nanopigments and the interactions between the dye and biomolecules from ULE. The presence of biomolecules seemed to have a complex effect, including the modification of crystalline structure, proven by the increase in basal spacing and the stability of the pigments. Thus, the reduction stability and photostability of nanopigments obtained in ULE were superior to that of nanopigments obtained in water, meaning an advantage in potential applications. The adsorption capacity of Bentone 34 is higher in ULE compared to water, leading to the possibility of obtaining nanopigments with a higher amount of MB and more stability. The adsorption study revealed that monolayer adsorption (Langmuir model) is most probable in water and multilayer adsorption is most probable in ULE (Freundlich model). In addition to the possibility of being applied as pigments, we propose the use of the composites for seed treatment, based on the presence of both clay and MB, a well-known sterilizer.