*4.2. Dyes*

Today, dyes are one of the main pollutants. They are produced in large quantities due to their widespread use, and for this reason, they can be released into natural environments. The release of these types of compounds causes serious problems to natural ecosystems due to their toxicity, carcinogenicity and because they also impart an intense color to the waters (even at very low concentrations) and, in addition, they are considered recalcitrant compounds. Therefore, e ffective treatments are necessary to remove these compounds from the waters. Biosorption is an excellent alternative to conventional methods. In fact, many papers have been published on the removal of these pollutants by biosorption, and recently, dyes continue to dominate biosorption applications.

The cationic dye methylene blue is one of the most widely used dyes in the industry, and for this reason, there is a wide variety of biosorbents that have been evaluated for the elimination of this dye. In recent years, there are also various examples of these proposals, in which both modified and unmodified biomass are used. Thus, coconut waste, chemically modified with acrylic and polyacrylic acids, has been utilized for the removal of this dye. With these modifications, the maximum sorption capacity reached was 138.88 mg/g at pH = 10.0 using the acrylic acid. The sorption mechanism is mainly based on electrostatic interaction and on Lewis acid-base interaction [30]. *Cortaderia selloana* flower spikes transformed into nanomagnetic particles reached a maximum removal capacity of this dye of 119.05 mg/g at pH = 6.0 and through electrostatic interactions [132].

However, unmodified biomass methods are still the most widely used to remove methylene blue. Recently, it has been made possible to find many examples of this. Brewer's spent grain has recently been used with a maximum adsorbed amount of methylene blue of 284.75 mg/g at pH = 11.0. A possible adsorption mechanism involves electrostatic interaction, electron donors and electron acceptors, hydrogen bonds and π-π dispersion [133]. Biomass obtained from weeds (*Cyanthilium cinereum* and *Paspalum maritimum*) was also evaluated to eliminate this dye, the maximum absorption capacities obtained were 56.18 and 76.34 mg/g, respectively [134]. *Fucus vesiculosus* dead biomass was also used to remove this dye, presenting a maximum biosorption capacity of 698.48 mg/g at pH = 6.0 by a physical biosorption mechanism related to a cation exchange process between the dye and biomass functional groups, releasing protons (H+) to the system [135]. Biomass from *Bifurcaria bifurcata* was also applied to remove this dye with a maximum biosorption capacity of 2744.5 mg/g in only 15 min. The best adsorption e fficiency was obtained at pH = 5.6 due to electrostatic interaction [136]. In these examples, although the biomasses of these macroalgae were clearly higher, it is necessary to consider that the brewer's spent grain is a byproduct of the brewing industry produced in large quantities and with few ecological disposal options; in addition, weeds are abundant and with few applications. This shows that the choice of a certain biosorbent depends on many factors that must be evaluated together. The search for new biosorbents is essential to achieve this objective. An interesting example of this is the application of the biomass from brazilian berry seeds (*Eugenia uniflora*) to remove this dye in conditions closer to reality using two di fferent simulated e ffluents with a color removal greater than 70%. This material had a biosorption capacity of 189.6 mg/g at pH = 8.0 and presents OH groups that can perform hydrogen and electrostatic bonds with methylene blue [56]. Other macroalgae also recently used as biomass were *Ulva fasciata* that reached a maximum adsorption capacity of 244 mg/g and *Sargassum dentifolium* with only 66.6 mg/g. In both cases, it was determined that the biosorption of methylene blue was independent of pH [12].

Crystal violet is another cationic dye that has recently received attention for biosorption. Powdered seeds of the araticum fruit (*Annona crassiflora*) were used in the biosorption of this dye with a maximum biosorption capacity of 300.96 mg/g in 120 min at pH = 7.5. Electrostatic interaction played an important role in the biosorption process of this dye since a pH higher than the zero charge point of the biosorbent (7.2) causes the surface of this material to be negatively charged due to its deprotonation, which favored an increase in its biosorption capacity because crystal violet is a cationic dye, and therefore, interaction with a negative charge on the surface increases the amount of dye biosorbed [18]. Biomass from *Diaporthe schini* (new fungus recently discovered) reached a maximum

biosorption capacity of 642.3 mg/g. This biomass was also evaluated in a simulated effluent with a considerable reduction in the color [137]. Finally, as a more sophisticated option, water dispersible Fe3O4/Chitosan/Glutaraldehyde nanocomposites (superparamagnetic) were also tested, in this case with a maximum biosorption capacity of 105.47 mg/g. Electrostatic interaction between the negative charge of the biosorbent surface and the positive charge of the dye would explain why this maximum capacity took place at pH 11.0. Although the maximum removal capacity was lower than the previous cases, the advantage of this technique is its ease and rapid separation from samples, allowing a reusability up to at least ten cycles [138].

The discarded seed biomass from pepper (*Capsicum annuum*) was tested to remove Basic red 46 dye. Taguchi DoE methodology was employed to optimize the process reaching a dye removal performance of 92.1 mg/g at pH = 8.0 [139].

Biomass derived from macroalgae was also evaluated to remove the Rhodamine B dye. The macroalgae used were *Kappaphycus alvarezii*, *Gracilaria salicornia* and *Gracilaria edulis*, both in native form and ethanol modified. The maximum biosorption capacity determined as 9.84, 11.03, 8.96, 112.35, 105.26 and 97.08 mg/g at pH = 2.0, respectively. At this low pH, there is an increase in the protonation effect on the surface of these materials resulting in a higher biosorption capacity. The modified biomass was more efficient [140].

Anionic dyes are the other group of dyes that also have a multitude of applications, and for this reason, they are also an environmental problem. These types of dyes have also recently been studied as applications in the field of biosorption. Thus, the removal of tartrazine yellow was evaluated using brewer's spent grain as biomass; the maximum adsorbed amount was 26.18 mg/g at pH = 2.0. This adsorption involves electrostatic attraction, π-π interaction and multilayer formation of dye [133]. Reactive Blue 19 using dead biomass of the brown marine alga *Bifurcaria bifurcata*, with a maximum adsorbed amount of 88.7 mg/g in only 15 min at pH = 1.0. At this very low pH, the concentration of H3O+ was high enough to allow the protonation of sulfonate groups of this dye, which favors the interaction between the dye and the functional groups of the biomass [136]. Eriochrome black T is another example of an anionic dye recently studied for its elimination by biosorption; in this case, using dead biomass of *Fucus vesiculosus* and with a maximum biosorption capacity of 24.31 mg/g at pH < 4.0. Van der Waals interaction was the main interaction mechanism between this dye and biomass [135]. Biomass from *Ocimum gratissimum* leaves was tested for the indigo carmine dye biosorption. This biomass obtained a maximum biosorption capacity of 77.52 mg/g, confirming that this capacity was superior to that of other sorbents used to remove this dye. Since this dye is anionic, the most favorable adsorption occurred at pH 2.0 because the surface of this biosorbent is positively charged [128]. Direct Fast Scarlet 4BS was successfully removed using dead biomass from *Enteromorpha prolifera* with a maximum sorption capacity of 318.87 mg/g also at pH 2.0. The adsorption mechanism involved hydrogen bonding, electrostatic attraction and bonding and hydrophobic and van der Waals interaction [127]. Finally, Reactive Red 120, using immobilized biomass of *Pseudomonas guariconensis* in a Ca-Ag biocarrier matrix, was efficiently eliminated. In this case, since the biomass was alive, in addition to biosorption, biodegradation occurred. Toxic reactive dye was converted into non-toxic compounds. The immobilized bacterial cells exhibited 87% uptake of this dye, whereas the non-immobilized bacterial cells exhibited a maximum uptake of 37% [6].

Anionic dyes are better adsorbed at low pHs—that is, at pHs below the zero-charge point of the biosorbent—because under this condition, the surface of the biosorbent acquires positive charge.

### *4.3. Other Organic Pollutants*

Other organic pollutants of interest have also been treated using biosorption as the primary removal technique. Table 3 shows some examples of the most recently studied organic pollutants.


**Table 3.** Examples of other organic pollutants that have recently been evaluated for their removal using biosorption.

As can be seen in this table, the nature of the organic compounds is very varied, which is indicative of the enormous possibilities that biosorption techniques have for the removal of this type of pollutant. In addition, these examples reflect the flexibility of biosorption techniques since, in the same way as for other pollutants, biomass can be alive or dead, in batch or in fixed-bed experiments, but it is noteworthy that even this technique can be coupled to an alternating current system that allows increasing the speed of biosorption [38]. However, the removal capacity that some of these sorbents have is far from that achieved with commercial sorbents such as activated carbon. Although the comparison data are scarce, the values obtained indicate the need to search for biomaterials with greater capacity—for example, the biomass of *Scenedesmus obliquus* had a maximum removal capacity of salicylic acid and ibuprofen of 63 and 11.9 mg/g, respectively; instead in the same conditions, activated carbon had 250 and 147 mg/g [144].

### **5. Conclusions and Future Perspectives**

As can be seen, the field of biosorption continues to offer very promising results for the elimination of pollutants. It is a technology that presents a grea<sup>t</sup> diversity of options and combinations, demonstrating grea<sup>t</sup> flexibility for its application. It is difficult to limit the studies that try to reveal the properties of any material for use in biosorption. The reason for this is obvious: the amount of possible materials (living or dead) is enormous. These studies must continue to progress because without a material with adequate properties, biosorption cannot be competitive. However, it is not only necessary to determine the properties of a possible material but also to evaluate it compared to others already established as sorbents (commercial sorbents), and therefore, conclude that this new material is a better alternative. There are still steps to be taken for biosorbents to be fully accepted. Currently, there are several challenges of biosorption: the development of large-scale procedures, greater commercialization and, in general, its application in real conditions. Although the advantages of this type of sorbent are evident (mainly cost), few biosorbents are currently marketed for their use [22]. The application of biosorption at the industrial scale has not been ye<sup>t</sup> well exploited, and this constitutes another of the weaknesses that biosorption must face. Still, the vast majority of biosorption applications focus on laboratory studies. All these studies make possible the current knowledge about biosorption that is enough to provide a solid base that allows its use to be extended. However, this process is not widely used in industry.

An important reason that can explain these weaknesses is that biosorbents, in their natural state, tend to have a lower removal capacity than traditional or conventional sorbents such as activated carbons, zeolites or ion exchange resins. However, it is difficult to ascertain this fact because at present there are still few studies in which a biosorbent is compared with commercial sorbents under the same conditions [145,146]. Perhaps, the fact of thinking that a biosorbent may be less effective than the traditional ones could be counterproductive to achieve that biosorbents climb positions, because the true capacity of these materials will remain unknown. The fact that the matrices used in the experiments have different physicochemical properties does not help much in improving the perception of biosorption, since this makes it difficult to compare biosorbents to obtain the one with the highest affinity for a pollutant. A certain standardization could be interesting to solve this aspect.

In any case, if the above is true and biosorbents lack the necessary efficiency, this would imply the need to modify them to achieve greater efficiency. It would be desirable if the biosorbents had, at least, characteristics comparable in efficiency to the commercial ones. There are several alternatives that can improve the effectiveness of biosorbents, ranging from chemical or physical modifications to the use of nanomaterials [29,63,73]. Chemical of physical modifications applies mainly to dead biomass. However, these alternatives would increase the cost of the final product, and the resulting material could be even less eco-friendly, reducing the virtues of biomaterials. In this context, it is interesting not to forget the use of living biomass to improve the effectiveness of a pollutant removal process. Despite the advantages attributed to dead biomass, the properties of living biomass for application as biosorbents have not ye<sup>t</sup> been adequately exploited or even better studied. Many studies indicate that the use of living biomass is more efficient than dead biomass, and living biomass is used without modification. The cost of the production and maintenance of living biomass is among the problems attributed to the use of this type of biomass; however, there are organisms that can be cultivated intensively and with low cost [147]. Macrophytes, microorganisms such as microalgae or some species of bacteria offer very promising results. Studies in this direction should continue.

Immobilization is another key mechanism to improve biosorption processes. It is also a fundamental mechanism for the application of biosorbents on an industrial scale. At present, different proposals are still being evaluated to solve the practical problems of immobilization, especially when living biomass is used [106]. In fact, there has been an increase in the number of studies using immobilized living biomass, perhaps because for many industrial applications, the use of living biomass is preferable. The support for this biomass is being increasingly perfected, as well as the search for the most suitable living biomass for each case, which is essential to ensure that a biosorbent can be successful in its application. An example of this improvement is an alternative that is being exploited by combining nanoparticles with biomass. Microbial cells immobilized on magnetic nanoparticles is a relevant new technique applied to obtain new biosorbents, which has several advantages [29]. However, it is necessary to recognize that the cost of these biocomposite materials can be uncompetitive, as well as an option that can be considered not very eco-friendly. In any case, living biomass immobilization techniques must continue to be refined, seeking more natural and cheaper supports.

Another weakness of biosorption is that many biosorption studies use synthetic wastewater or solutions in distilled water, which does not take into account the behavior of these sorbents with different competitors or with physicochemical parameters that can differ considerably in real conditions. The evaluation of biosorbents in real situations would provide more information and would allow a more adequate assessment of the possibilities of this technique. For this reason, future research should be directed in this direction.

Finally, it is hoped that in the future, as the weaknesses are resolved, biosorption will find its place in industry and in separation technologies.

**Funding:** This work was carried out with the financial support of the Spanish "Ministerio de Economía, Industria y Competitividad" (CTM2017-88668-R).

**Conflicts of Interest:** The authors declare no conflict of interest.
