Introduction

Dyes are considered as one of the important pollutants in aquatic environments due to their negative effects. Among these effects, the toxicity to living beings, and the coloration of the waters are the most notable. Millions of tonnes of dyes are currently produced per year, and this production is increasing to supply the enormous demand for these compounds by industries. The textile industry, paints and coatings, construction, plastics are the main applicants for dyes (Tkaczyk et al. 2020). As a result of this enormous use, a significant amount of dyes are discarded through effluents without reaching an adequate degree of treatment and they can reach aquatic environments with very negative consequences.

The Brilliant green dye can be classified as one of the dyes considered as priority because in recent years its use has increased in multiple industrial sectors due to its easy and cheap production (Singh et al. 2016). Currently, it is widely used in the manufacture of inks for printing on paper, in staining compounds in the textile industry, in staining biological samples for clinical analysis, in culture media for microbiology, in veterinary medicine, as a disinfectant, as an inhibitor of molds or of propagation of fungi, in cosmetics, and also in the food industry (Sabnis 2010). However, the presence of this dye in natural environments is related to health disorders and environmental impact. This dye is known to cause serious eye irritation, skin and respiratory tract irritation, causing coughing and shortness of breath, as well as gastrointestinal tract irritation (Ragab et al. 2019; Giri et al. 2020; Melhaoui et al. 2021), problems to which must be added that many of the possible adverse effects on human health are still unknown (Melhaoui et al. 2021). In fact, this dye is related to carcinogenic and genotoxic properties (Oplatowska et al. 2011). For these reasons, possible solutions must be sought for its elimination from natural environments, solutions that must be efficient, economical, and respectful with the environment.

The decolorization and detoxification of natural environments polluted with dyes is one of the most important aspects to regenerate these systems and is a major concern to meet environmental regulations. At present there are different treatment methods to eliminate dyes such as coagulation-flocculation, oxidation, ozonation, nanocomposites (Bonyadi et al. 2022a, b; Islam et al. 2023). However, these methods can be ineffective, expensive, or have unwanted effects. These problems can be avoided by using other more environmentally friendly methods such as biosorption or biodegradation (Torres 2020; Zahmatkesh Anbarani et al. 2023). These methods use biomass of different origin to eliminate pollutants from natural environments. Biosorption has been used successfully for a long time to remove dyes (Rusu et al. 2021; Nasoudari et al. 2023). Biomass has excellent properties for removing various pollutants, especially those that may be charged, such as many dyes. This is because biomass is made up of very diverse materials that provide various functional groups. For example, for the elimination of cationic dyes, such as brilliant green, which are positively charged, functional groups such as hydroxyl and carbonyl are important because they serve to establish electrostatic interactions (Nasoudari et al. 2023). In addition, these materials offer other possibilities of interaction with pollutants such as hydrogen bonding, cationic exchange, and pi interaction (Gul et al. 2023). For this reason, biosorption is an efficient methodology to eliminate pollutants.

The marine environment receives large amounts of pollutants from various sources, which also requires appropriate solutions to remove these pollutants (Willis et al. 2022). However, the assays in the search for solutions to eliminate pollutants in seawater are scarce. For these reasons, the objective of this work is to test the living biomass (without any modification) of the marine microalga Phaeodactylum tricornutum to remove Brilliant green from seawater solutions. This biomass has shown to have good properties to remove other pollutants present in seawater (Santaeufemia et al. 2016, 2019), including other dyes (Santaeufemia et al. 2021). Knowing the ability of this biomass to remove the brilliant green dye can be useful for the application of this biomass in biotechnological processes.

Material and methods

Biomass

The biomass used in this study was obtained from a stock culture of the marine microalga (diatom) Phaeodactylum tricornutum Bohlin (CCAP 1055/1). This alga was cultured using seawater enriched with ALGAL medium, and kept in a culture chamber at 18 ± 2 ºC with artificial light of an intensity of 68 μmol photons m-2 s-1 and 12:12 h of light/dark. The cultures were aerated a a volumetric rate of 10 L min-1 air. The biomass used for this work was living biomass taken directly from this stock culture.

Brilliant green dye

Brilliant green dye (CAS 633-03-4; C.I. 42040) of chemical formula C27H34N2O4S, and molecular structure shown in Fig. 1 is a cationic dye that belongs to the group of triphenyl methane dyes. This dye was obtained from Merck (Germany). Two stock solutions, 1 and 5 g L-1, were prepared in deionized water. Adequate volumes were taken from these solutions to obtain the concentrations used in the experiments. These concentrations were: 5, 10, 20, 25, 50, 75, 100, 150 and 200 mg L-1.

Fig. 1
figure 1

Structure of a) Brilliant green and b) leuco-Brilliant green

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Seawater

The seawater used in the experiments was natural seawater filtered through 0.22 μm pore size filters and sterilized in autoclave (121°C, 1 atm above standard pressure, 20 min). The salinity was 35 ‰ and the pH was 8.2 ± 0.1, adjusted with HCl or NaOH when necessary.

Experimental methods

Biosorption assays

The biosorption assays were carried out under axenic conditions for 7 h in glass tubes (Kimax) that were incubated at 18 ± 2 ºC, with artificial light at intensity of 68 μmol photons m-2 s-1 and subjected to gentle agitation (200 rpm) using an orbital shaker to ensure homogeneity.

Three replicates of each of the following elements were prepared for each concentration tested of the dye: control tubes without biomass exposed to light and without exposed to light (the latter covered with aluminum foil), and tubes exposed to light containing the biomass. A suitable volume of the dye stock was added to each tube containing the sterile seawater to obtain the final concentrations indicated above for carrying out each experiment. Then, in the tubes that should have the biomass, an adequate volume of the stock culture of the microalgae was added. The biomass concentration used in all experiments was equivalent to 0.4 g L-1 dry biomass. To obtain the volume data that was necessary to take from the stock culture to achieve this amount of biomass, the microalgal cells were counted under a microscope using a Neubauer chamber to obtain the cell density of this stock culture. For the final calculation of the necessary volume, the cell dry weight of this microalga was considered. Finally, all tubes were filled with sterile seawater to a final volume of 50 mL.

The tubes were incubated with the conditions indicated above, and sampled at different times: 0, 0.083, 0.25, 1, 2, 3, 4, 5, 6, and 7 h. The samples were centrifuged at 13000 xg for 2 min to obtain the supernatants, which were promptly measured.

Study of the effect of pH

To assess the effect of pH on the dye removal capacity, control tubes and tubes with biomass were prepared in the same way as in the previous section but, with the difference that the seawater was adjusted to different pHs with HCl or NaOH. In this study, the effect of the pH range 4 – 10 was verified. The dye concentration used for these experiments was 100 mg L-1.

Analytical methods

Measurement of dye concentration

Two different techniques were used to determine the concentration of the dye in seawater over time:

- A spectrophotometric measurement on a PharmaSpec UV-1700 UV/Vis spectrophotometer at 630 and 300 nm. Different concentrations of Brilliant green dissolved in seawater at pH 8.2, measured at the moment of its dilution from the stock solution, were used to obtain a calibration curve. The obtained equation that related these wavelengths with the concentration of the dye in seawater at pH 8.2 was:

$$\begin{array}{cc}\left[Brilliant\; green\right](mg\ {L}^{-1})=\;0.02+10.49*{A}_{630}+19.52*{A}_{300}& {{\text{r}}}_{{\text{adj}}}^{2}=0.991\end{array}$$
(1)

This measurement was used to determine the evolution of discoloration by quantifying the loss of color.

- A measurement by HPLC using a HP 1040 series equipment with UV/Vis detector and C18 column (250 × 4.6 mm). The mobile phase was a mixture of 0.05 M ammonium formate pH=4.0 (mobile phase A) and acetonitrile (mobile phase B). The gradient of elution was: 85 % A for 2.5 min, then from 15 % B to 100 % B in 10 min. A flow rate of 1 mL min-1 at 25ºC, 20 µL of injection, and detection at 300 nm were used. Brilliant green was used as standard.

The percentages of discoloration and dye removed at time t were calculated using the equation:

$${P}_{t}=\frac{\left({C}_{i}-{C}_{t}\right)\times 100}{{C}_{i}}$$
(2)

where Ci and Ct are the initial concentration of the dye and the concentration in the tubes with biomass and light at time t (mg L-1), respectively, measured either by spectrophotometry or by HPLC.

The amount of dye removed per unit of biomass at time t (qt) (mg g-1) was calculated as:

$${q}_{t}=\frac{\left({C}_{i}-{C}_{t}\right)\times V}{m}$$
(3)

where Ci is the initial concentration of the dye in solution measured by HPLC (mg L-1), Ct the concentration at time t (mg L-1), V is the volume of the solution (L), and m is the mass of the sorbent (g).

Kinetic study

The kinetics describe the evolution of the amount of dye removed over time, and the kinetic parameters make it possible to predict the rate of removal. This, in turn, provides important information for designing and modeling the process. In this study, the kinetic models shown in the Table 1 were tested to establish which of them best described the process.

Table 1 Kinetic and isotherm models included in this study
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Determination of Equilibrium Isotherms

To describe the properties of the biomass in relation to its Brilliant green removal efficiency, the isotherm models shown in the Table 1 were used. These models were used to analyze the data obtained from the equilibria.

FTIR

In order to identify functional groups on the biosorbent, Fourier transform infrared spectroscopy (FTIR) was used. FTIR spectra were obtained on a FTIR spectrometer (Thermo Scientific Nicolet iS10) using the attenuated total reflection (ATR) mode, with a resolution of 4 cm-1 and 128 cumulative scans. The range studied was 4000–500 cm-1. The microalgal biomass, before and after the biosorption, was dried and ground before analysis.

Statistical analysis

All data represent the mean of three replicates ± standard deviation. The data from the different experiments were fitted to the kinetic and isotherm equations using a nonlinear regression analysis. To assess the goodness of the fits, the r2adj coefficient was used. One-way ANOVA and Tukey's test, and two-way ANOVA were used, when necessary, to compare the means of the different treatments (α = 0.05). The data were previously checked to ensure that they met the postulates necessary to apply these analyses. Statistical analyses were performed with SPSS 27 (IBM, Spain).

Results

Biomass characterization

The chemical properties of the biomass surface are important to obtain an efficient biosorbent, and to study the mechanism of sorption. The FTIR spectrum allows to identify groups involved in the biosorption process. The infrared spectra obtained before and after the sorption process are shown in Fig. 2. The peak observed at 1045 corresponds to a stretching of Si-O-Si and is characteristic of diatoms. The region from 1000 to 1260 corresponds to carbohydrates. The bands from 1650 to 1735 are due to the stretching of C=O bonds and the N-H bending vibration. These bands correspond to the so-called protein-associated Amida I and Amida II bands. Peaks around 2970 reflect asymmetric and symmetric C-H stretching modes. The peak around 3279 corresponds to the O-H stretching vibrations. The presence of these functional groups is a good indication of the good properties of this biomass for the biosorption of this dye.

Fig. 2
figure 2

FTIR spectra of P. tricornutum biomass before and after Brilliant green removal. The arrows indicate the main differences between the two spectra

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After sorption, the main differences between both spectra were a new band around 1580 that is related to the vibration of C=C stretching of the aromatic group of the dye, and the region of 1100-1400 that appeared very different. These differences indicate that the dye was removed by the biomass.

Brilliant green stability in seawater without and with exposure to light

The results obtained with the control groups of experiments indicated that this dye was not stable in seawater, which is observed as a discoloration throughout the exposure time. Figure 3 shows the concentration of the dye measured by spectrophotometry. This measurement is indicative of the evolution of the color in the different concentrations of the dye in seawater, in the dark, during the 7 h of the experiments. As can be seen in this figure, the coloration decreased over time in all the concentrations tested. The data obtained could not be adequately fitted to conventional kinetic models (fits not shown). However, pseudo kinetic models were more suitable for this process. The fits with these kinetic models indicated that the discoloration followed a pseudo-second order kinetics (radj2 = 0.986 - 0.997) whose parameters are shown in Table 2. Between 47-65% of the color was lost after 7 h of exposure depending on the initial concentration of the dye.

Fig. 3
figure 3

Evolution of the concentration of the Brilliant green dye measured spectrophotometrically (discoloration) in control cultures with seawater at pH 8.2 in the dark and without biomass. Points are mean values ± SD (n=3)

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Table 2 Fits to pseudo-order kinetic models of the data obtained from the amount of Brilliant green dye lost due to spontaneous discoloration, in darkness and without the presence of biomass, after 7 h
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In addition to seawater, the experiments were carried out with lighting to keep the living microalgal biomass active. Since many dyes undergo photodegradation, this effect could contribute to further color reduction. To determine the contribution of the effect of light to the loss of color and to assess the possible photodegradation of the dye, control experiments were established as in the previous case, but exposed to the light intensity used in the experiments with biomass. The results obtained indicated the non-existence of photodegradation. Statistical analysis using a two-way ANOVA (illumination and initial dye concentration as factors) for all dye concentrations tested after 7 h of exposure showed that there were no significant differences between light and no light exposed controls (F1,36 = 0.76, p = 0.389). The interaction between these two factors was also non-significant (F8,36 = 1.07, p = 0.405). Therefore, the lighting conditions used in the experiments did not significantly contribute to the discoloration of the dye at the concentrations tested.

This result of spontaneous discoloration in seawater could be interpreted as an apparent removal of the dye (for example, by any biomass present in a biosorption test). However, even if the color disappears, the dye may persist in the medium, although in a different chemical form. Figure 4 shows the comparison between the dye concentration (5 mg L-1) measured by spectrophotometry and measured by HPLC, over time. It can be seen that the concentration of the dye remains stable over time when measured by HPLC. In contrast, the spectrophotometric measurement indicates an apparent loss of dye, although this loss only implies discoloration.

Fig. 4
figure 4

Evolution of the concentration of Brilliant green (5 mg L-1) in seawater at pH=8.2 measured by spectrophotometry, and by HPLC. Points are mean values ± SD (n=3)

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Biomass effect on discoloration

Figure 5 shows the evolution of discoloration in cultures exposed to light, but with biomass of the microalga. As can be seen in the figure, despite the spontaneous discoloration of the dye, discoloration was faster and more effective than in the absence of biomass (see Fig. 3 for comparison). After 7 h of culture, 96.3 ± 0.6 % of the color was eliminated in the cultures with the highest concentration of the dye (200 mg L-1) compared to 65.3 ± 1.9 % of the assays without biomass. With the presence of biomass, almost 100 % of the color was removed at initial dye concentrations ≤ 100 mg L-1 during this time. Statistical analysis by two-way ANOVA (presence of biomass and dye concentration as factors) indicated that both the effect of biomass (F1,36 = 91443.11, p<0.001) and concentration (F8,36 = 9017.23, p<0.001) were statistically significant. In addition, the interaction between these factors (F8,36 = 6604.29, p<0.001) was also statistically significant.

Fig. 5
figure 5

Evolution of Brilliant green discoloration in cultures with microalgal biomass. Points are mean values ± SD (n=3)

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Characterization of the biosorption process

The measurement of the concentration of the dye by HPLC allowed the determination of the amount of dye that was truly removed by the microalgal biomass, because this measurement is not based on the loss of color that this dye experiences in natural seawater, remaining in the medium, although without color.

Effect of biomass on the amount of removed dye

Figure 6a shows the evolution of the amount of removed dye determined by HPLC throughout the culture time. As can be seen, the concentration of the dye in seawater decreased over time, which indicates that the biomass of this microalga was able to remove this dye. There was an initial period of rapid removal until equilibrium was reached, then the concentration of removed dye remained constant. This equilibrium was achieved during the duration of the experiment, 7 h. In fact, only 3 h were necessary to reach the equilibria. Therefore, a duration of the experiments of 7 h was a sufficient time to characterize the removal of the dye by this biomass. Up to the concentration of 10 mg L-1 of dye, this biomass was able to remove 100 % after 7 h of process. At the highest concentration of dye (200 mg L-1), the amount removed was 32.6 ± 0.3 %. The two-way ANOVA (initial dye concentration and presence of biomass as factors) indicated that the dye concentration measured by HPLC after 7 h of processing showed significant differences for both factors. The amount of dye removed depended on the initial dye concentration (F8,36 = 4000.40, p<0.001) and the presence of biomass (F1,36 = 4103.94, p<0.001). It is important to note that the interaction between both factors was also significant (F8,36 = 180.38, p<0.001). The interaction plot for the biomass effect shows that this interaction occurred at the lowest dye concentrations.

Fig. 6
figure 6

a) Evolution of the concentration of Brilliant green measured by HPLC (removal) in cultures with microalgal biomass. Points are mean values ± SD (n=3). b) Interactions plot derived from the two-way ANOVA for the effect of biomass at 7 h of processing

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The amount of dye removed per unit of biomass in the different concentrations tested is shown in Fig. 7. As the initial concentration of the dye increased, the final amount removed per unit of biomass at 7 h increased up to the concentration of 150 mg L-1. From this concentration, the amount of removed dye remained constant due to the saturation of the biomass. The dynamics of this process was calculated using kinetic models. Thus, in order to determine the kinetic characteristics that followed the removal process, two kinetic models (pseudo-first and pseudo-second order) were tested. The analysis indicated that the best fit of the data was with pseudo-first order kinetics. The parameters obtained with this fit are shown in Table 3. The mean removal time (t1/2) was calculated using the formula:

$${t}_{1/2}= {~}^{Ln 2}\!\left/ \!{~}_{{k}_{1}}\right.$$
(4)

where k1 is the pseudo-first order kinetic constant obtained from the fit to this kinetic model. This time was less than 0.75 h, even at the highest concentrations of the dye. The maximum dye content reached by this biomass was high, with 160.78 ± 1.47 mg g-1. Therefore, the removal process of this dye by the microalgal biomass was fast, indicating that there was a high affinity of this biomass for the dye.

Fig. 7
figure 7

Evolution of the concentration of Brilliant green removed per unit of biomass. Lines obtained with a fit to pseudo-first order kinetics. Points are mean values ± SD (n=3)

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Table 3 Kinetic parameters for the removal of the Brilliant green dye by the biomass of the microalga P. tricornutum
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Isotherm studies

Data obtained from equilibrium concentrations of this dye in seawater and biomass are essential to understand the process. Figure 8 shows the data obtained in equilibrium for the dye concentrations tested and the fit of the data to the isotherm models used. The parameters obtained with these models are shown in Table 4. The isotherm model that obtained the best value of the correlation coefficient (radj2) was the Langmuir's model. From this model, a maximum biosorption capacity (qmax) of 161.52 ± 5.95 mg g-1 was obtained. The values obtained for the separation factor (RL) derived from this model, for all concentrations of Brilliant green tested, were from 0.009 to 0.28. The isotherm model that obtained the worst value of radj2 was the Freundlich model. However, this value was also high, so the information derived from the parameters can be considered significant to understand the biosorption process. The KF parameter is related to the affinity of the biomass for the sorbate; the value obtained, 65.32 ± 6.86 mg1−(1/n) L1/n g-1, was high, as will be discussed later. The rest of the isotherm models are related to the energy of the biosorption process, indicating that it was an endothermic, favorable, and spontaneous process.

Fig. 8
figure 8

Equilibrium isotherms for the removal of Brilliant green by the biomass of the microalga P. tricornutum. qe and Ce are the amount of dye removed per unit of biomass, and the residual concentration of dye in the medium, respectively, both at equilibrium

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Table 4 Parameters derived from the isotherm models used in this study, and the error function (radj2) used to assess the goodness of fit
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Effect of pH

The initial pH of the solution is one of the factors that has a great influence on the sorption processes of charged compounds since it influences the distribution of charges of the sorbent and sorbate. The assays were performed in a pH range of 4-10, after verifying that the concentration of this dye, measured by HPLC, remained stable in this pH range in the absence of biomass. The results of the assays are shown in Fig. 9. As can be seen the percentage of dye removed increased as the pH increased until reaching a pH of around 7, where the removal percentage was 60.9 ± 2 % for an initial Brilliant green concentration of 100 mg L-1. Statistical analysis by one-way ANOVA and Tukey's test indicated that this increase was statistically significant (F6,14 = 133.21, p<0.001). From this pH, the percentage removed remained without significant differences. A significant increase between pH 6 and pH 7 is also shown in this figure.

Fig. 9
figure 9

Effect of pH on the percentage of Brilliant green removal by P. tricornutum biomass using a concentration of 100 mg L-1 of dye. Different letters indicate significant differences (p<0.05)

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Kinetic model for Brilliant green discoloration using biomass of the microalga P. tricornutum

Kinetic studies are important to determine the speed of the process, and it is a first approximation to understand the mechanisms involved. For this reason, it is necessary to know the kinetics of discoloration in the presence of microalgal biomass. In order to determine the global kinetics of this process, the data obtained were adjusted to a general pseudo-order kinetics since in this study it was observed that the discoloration was a complex process due to two mechanisms, the chemical transformation of the dye in the seawater, and the removal by the biomass. This adjustment allowed determination of the global pseudo-order of the reaction that was obtained with the experimental data. The equation of the general pseudo-order kinetic model is shown in Table 1. The results obtained from the adjustments to this model are shown in Table 5. As can be seen in this Table, the pseudo-order of the reaction (n) increased with the initial concentration of the dye. The pseudo-order was 1 at the lowest concentrations, reaching a third-pseudo-order kinetics value at the highest concentration tested. With the data of the kinetic parameters obtained, the average discoloration time was calculated using the equation:

$${t}_{1/2}=\frac{{2}^{n-1}-1}{{K}_{n}*{C}_{re}^{n-1}*(n-1)};\; n\ne 1$$
(5)

where Kn is the kinetic constant, n is the order of the reaction, and Cre is the concentration of dye removed at equilibrium. These parameters were those obtained from the kinetic adjustment. The average discoloration time obtained was not greater than 0.45 h, which indicates that the discoloration was rapid and inversely proportional to the initial concentration of the dye.

Table 5 Parameters obtained from the adjustment to general pseudo-order kinetics for the decolorization process of the Brilliant green dye in seawater with the presence of P. tricornutum biomass
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Discussion

Seawater is a more complex solution than the usual solutions for biosorption assays with distilled water. For this reason, to correctly determine the properties of the biomass of the microalga P. tricornutum as a Brilliant green biosorbent, it is necessary to verify the stability of this dye in this medium and under the test conditions because abiotic transformation can contribute to increasing the amount removed, overestimating the true capacity of the biomass. The results shown in Fig. 3 indicate that this dye underwent spontaneous discoloration in seawater. It was verified that this process followed pseudo-second order kinetics in this medium. One of the reasons that can cause this decrease is that in aqueous solutions, this dye can be in two forms: the colored cationic form, and the uncolored carbinol form (Fig. 1). The dominant presence of one form or another is greatly affected by the pH of the solution due to its extended conjugated system of alternate double and single bonds. An increase in pH above 6 results in a progressive transformation to the colorless form (Leuco-Brilliant green) (Rao et al. 2016). Since the experiments were carried out in seawater at pH 8.2, the color reduction could be due to this effect.

On the other hand, it is also necessary to take into account that another possible cause of degradation of dyes is the effect of light, and as the experiments with living microalgae were carried out with illumination, it was determined whether this effect contributed to an additional discoloration. However, the results indicated that under the culture conditions used in the experiments this effect was not significant; therefore, the main cause of Brilliant green discoloration in seawater could be attributed to pH.

As a consequence of what was indicated above, in biosorption studies for the elimination of dyes, the measurement of the dye concentration using colorimetric data (spectrophotometric measurement) as analytical technique can lead to error, overestimating the amount truly eliminated since the dye remains in the medium, although in its discolored form. Spectrophotometric measurements are the most common to determine dye concentration, especially in biosorption assays, however, caution is necessary. For this reason, the chosen measurement parameter must be verified in relation to its stability for the test conditions. A clear example of this problem is this study, using biosorption techniques, for the removal of brilliant green dye in seawater solutions. The measurement of the dye by HPLC is a more reliable and accurate measurement than the colorimetric measurement by spectrophotometry (Fig. 4). Obviously, to previously avoid this problem, the pH of the seawater could have been adjusted to a suitable value to keep the color of the dye stable (pH = 6 or lower). However, this would not be a real situation because it is generally considered that the pH of seawater remains slightly alkaline (7.5 - 8.5). Therefore, for decontamination studies with this dye in seawater (or alkaline solutions) it is necessary to consider this effect. These results indicate that, in some cases, it is necessary to consider as different processes the discoloration and the true elimination of a dye.

It is evident that this dye experienced discoloration in seawater, however, the presence of microalgal biomass clearly accelerated the discoloration process. Therefore, in this case, the discoloration was due to the sum of two processes, spontaneous discoloration and biosorption by the microalga. The biosorption process followed a pseudo-first order kinetics, reaching equilibrium within 3 h (Fig. 7). The information obtained from sorption equilibria is essential to properly understand the biosorption process. Although there is a great diversity of isotherm models proposed, Langmuir, Freundlich, Temkin and Dubinin-Radushkevich are the most commonly used to characterize this type of processes. Although all models had a high correlation coefficient, the Langmuir model was slightly superior (Table 4). From this model it was deduced that the sorption of Brilliant green by the biomass of P. tricornutum was favorable for all concentrations tested. Since RL values lower than 1 were obtained from this model, the removal of this dye by the biomass can be considered favorable considering this criterion (Hall et al. 1966). From the other isotherm models, interesting properties of this biomass can be deduced. Thus, the parameter KF is derived from the Freundlich isotherm. The KF parameter is related to the affinity of this biomass for the dye. Since the value obtained was quite high (65.32 ± 6.86 mg1−(1/n) L1/n g-1), the affinity for this dye was high. Since a high affinity is a desirable value for an efficient biosorption process, this biomass offers good expectations for the removal of this dye. In addition to this parameter, the parameter 1/n gives information about the heterogeneity of the sorbent, and about the intensity of the sorption. The smaller this parameter is in relation to unity, the greater the sorption intensity, and values between 0.2 and 0.8 indicate greater heterogeneity. The greater the heterogeneity of the surface of the particle, the greater the surface area and the more suitable it is for biosorption of pollutants (Ighalo and Adeniyi 2020). The biomass of this microalga obtained a value of 0.21 ± 0.03, therefore, it has suitable properties to be used in biosorption processes.

The Tenkim isotherm model allows calculating the heat of the process. Since the value of the parameter bT was positive, the process can be considered exothermic. Finally, the D-R model can be used to calculate the free energy and interpret the biosorption process involved. Since the Ed value obtained for this biomass was high, the biosorption process of Brilliant green on this biomass would be mainly chemical.

The effect of pH is one of the main factors affecting biosorption processes. In fact, the removal efficiency of this dye by the living biomass of P. tricornutum varied as a function of pH. This efficiency was higher at pH values ≥ 7 (Fig. 9). This behavior can be explained taking into account the zero-charge point (pHpzc) of this biomass. This is an important parameter of the biomasses to study the biosorption of charged compounds because the solution pH affects the dissociation equilibrium of functional groups present in the biomass. The pHpzc of this biomass was previously determined, the value was 9.9 ± 0.03 (Santaeufemia et al. 2021). Considering this value, the number of positive charges of this biomass decreases as the pH approaches 9.9. As a consequence of this, the repulsion between the positively charged surface and the dye (with positive charge) is less and less. From that pH, the microalgal biomass acquires a negative charge, which would facilitate the union with the positively charged dye. However, with this dye it is necessary to consider an additional factor, starting at pH 6, this dye loses its positive charge as the pH increases, remaining uncharged. For this reason, subsequent increases in pH did not cause an increase in the percentage removed. In fact, from pH 7 onwards, the percentage of dye removed did not increase significantly. However, this uncharged form of the dye at pH > 6 could be the cause of the strong increase observed between pH 6 and 7 (Fig. 9). This uncharged form could be more easily transferred through the microalgal membrane, increasing the amount of dye removed, facilitating bioaccumulation. This result agrees with other results obtained, such as the high free energy of sorption, or the variation obtained in the FTIR spectra. Since the pH of the experiments was 8.2, it might be reasonable to think that the mechanism of Brilliant green removal by the living biomass of the microalga would not be solely electrostatic. The use of living biomass can increase the removal efficiency because this removal will not depend only on the adsorption of the dye, but bioaccumulation and biotransformation processes can also contribute. This hypothesis is reinforced by the fact that the statistical analysis showed a significant interaction between dye concentration and the presence of live biomass on both the decolorization process and the amount of dye removed (Fig. 6b). Thus, lower concentrations of the dye removed more dye than expected, which could be due to the fact that, at these low concentrations, the microalgal biomass remained more active (less possible toxic effect of the dye), facilitating the alternative processes indicated above. There is ample evidence to indicate that living biomass can be more efficient in biosorption than dead biomass (Isik 2008; Xu et al. 2020; Santaeufemia et al. 2021).

Mechanisms of biosorption of Brilliant green by living cells of P. tricornutum

The use of living cells constitutes a complex removal system in which the removal of the dye can be achieved by bioadsorption, bioaccumulation or even by biotransformation. In fact, living systems can be more efficient because the variety of processes involved is higher (Torres 2020; Zahmatkesh Anbarani et al. 2023). As discussed above, this could be one of the reasons why this biomass was so efficient in removing this dye, with a high amount removed per unit of biomass. Furthermore, biomass derived from microalgae is considered very suitable to be used in biosorption processes due to its surface properties. A wide variety of functional groups (as deduced from the FTIR spectrum) are available to capture the molecules of this dye through most interactions, cationic exchange, pi interactions, electrostatic attractions, hydrogen bonds. These functional groups are responsible for making the surface of the microalgae negatively charged. Of special relevance in the biosorption mechanism of this dye seem to be the electrostatic interactions as deduced from the clear effect that the pH had on the removal efficiency. This may be another reason for the high effectiveness of this biomass because it facilitates the binding of positively charged compounds such as brilliant green. It is evident that biosorption contributed to the discoloration process. This discoloration was faster and more intense in the presence of biomass. For this reason, it can be demonstrated that the Brilliant green discoloration process in seawater was the result of a synergistic effect between spontaneous discoloration and the effect of living microalgal biomass.

Comparison with other sorbents

Table 6 shows the capacity of other sorbents tested for the removal of Brilliant green. As can be seen, the living biomass of this microalga can be included in sorbents with a high capacity to remove this dye. It is important to highlight that many of these sorbents are processed or transformed materials, unlike the biomass of this microalga, which is natural and without transformation. This is an advantage, since only production by culture of the microalga is necessary to obtain this biomass. Currently, the culture of microalgae is simple and economical, so this biomass offers good possibilities for its application in biosorption processes, not only for the elimination of this dye but for other pollutants. The bibliographic data indicate that the biomass of this microalga was also efficient in removing other pollutants (Santaeufemia et al. 2016, 2019, 2021; Seoane et al. 2022). Furthermore, microalgal biomass is easy to manipulate and can be applied in biotechnological processes (Jacob-Lopes et al. 2020).

Table 6 Comparison of different sorbents used for the removal of Brilliant green
Full size table

Conclusion

Biosorption is an efficient, cheap, and environmentally friendly pollutant removal technique. However, to demonstrate that a certain biomass is suitable for this process, the tests must be correct. Verifying that the measurement used to determine the concentration of the pollutant is stable during the experiments is essential to correctly assess the properties of the biomass without making errors. An example is the Brilliant green dye in seawater that undergoes spontaneous discoloration, and therefore a colorimetric measurement with a spectrophotometer would not be correct to evaluate biosorption. Taking this into account, the results indicated that the living biomass of the microalga P. tricornutum without any type of modification could be very useful in the elimination of Brilliant green from seawater using the biosorption technique. It is an efficient biomass with high affinity for this dye.