**3. Biosorption of metals using** *Moringa oleifera*

Since *Moringa oleifera* seeds have the ability to retain metals, it is necessary to define and to understand the functional groups responsible for the adsorption phenomenon. Biosorption by dead biomass or by some molecules and/or their active groups is a passive process based mainly on the affinity between the biosorbent and the sorbate. In this case, the metal is sequestered by chemical sites naturally present in the biomass. The diagram in Figure 5 illustrates the main steps in this process. In most cases, the biosorption process is rapid and takes place under normal temperature and pressure. After the process of phase separation a biomass "charged" with metal ions and an effluent free of contamination are obtained. Two paths can be followed to deal with the "contaminated" biomass, the one of greatest interest being biosorbent regeneration and metal recovery. This process is the most attractive because biomass can be used for the removal of other metal species from other contaminated effluents. The other option is the destruction of the biomass, which offers no possibility of reuse.

**Figure 5.** Main steps in biosorption process [27].

coagulant chemicals, *Moringa oleifera* has a number of advantages including low cost, biode‐ gradable sludge production and lower sludge volume, and also it does not affect the pH of the water. Apart from turbidity removal, *M. oleifera* seeds also possess antimicrobial properties [24, 25], although the mechanism by which seeds act upon microorganisms is not yet fully

Tissues of *M. oleifera* from a wide variety of sources have been analyzed for glucosinolates and phenolics (flavonoids, anthocyanins, proanthocyanidins, and cinnamates). *M. oleifera* seeds reportedly contain 4-(α-L-rhamnopyranosyloxy)-benzylglucosinolate in high concentrations. Roots of *M. oleifera* have high concentrations of both 4-(α-L-rhamnopyranosyloxy)-benzylglu‐ cosinolate and benzyl glucosinolate. Leaves contain 4-(α-L-rhamnopyranosyloxy)-benzylglu‐ cosinolate and three monoacetyl isomers of this glucosinolate and only 4-(α-Lrhamnopyranosyloxy)-benzylglucosinolate has been detected in *M. oleifera* bark tissue [26]. Every glucosinolate contains a central carbon atom which is bonded to the thioglucose group (forming a sulfated ketoxime) via a sulfur atom and to a sulfate group via a nitrogen atom. These functional groups containing sulfur and nitrogen are good metal sequesters from aqueous solution. The leaves of *M. oleifera* reportedly contain quercetin-3-*O*-glucoside and quercetin-3-*O*-(6' '-malonyl-glucoside), and lower amounts of kaempferol-3-*O*-glucoside and kaempferol-3-*O*-(6' '-malonyl-glucoside), along with 3-caffeoylquinic acid and 5-caffeoylquin‐ ic acid. Neither proanthocyanidins nor anthocyanins have been detected in any of the tissues [26]. Although *M. oleifera* seeds have been most widely applied as a coagulant agent, many studies have been performed in order to explore other potential applications of this material,

especially in the removal of metals from aqueous systems.

understood.

**Figure 4.** Seeds of *Moringa oleifera* [18]*.*

230 Applied Bioremediation - Active and Passive Approaches

The mechanisms associated with heavy metal biosorption by biomass are still not clear; however, it is important to note that this process is not based on a single mechanism. Since metals may be present in the aquatic environment in dissolved or particulate forms, they can be dissolved as free hydrated ions or as complex ions chelated with inorganic ligands, such as hydroxide, chloride or carbonate, or they may be complexed with organic ligands such as amines, humic or fulvic acids and proteins. Metal sequestration occurs through complex mechanisms, including ion-exchange and complexation, and it is quite possible that at least some of these mechanisms act simultaneously to varying degrees depending on the biomass, the metal ion and the solution environment.

In reference [28] indicated that ion-exchange is an important concept in biosorption, because it explains many of the observations made during heavy metal uptake experiments. In this context, the term ion-exchange does not explicitly identify the mechanism of heavy metal binding to biomass, and electrostatic or London–van der Waals forces should be considered as the precise mechanism of chemical binding, i.e., ionic and covalent bonds. Figure 6 provides a schematic representation of an ion-exchange mechanism for a biosorbent material where "Me" represents a metal with valence +2.

component that appears due to the presence of lipids can be seen at 1740 and 1715 cm-1, as can be observed in the infrared spectra as small peaks, and the shoulders forming part of the main band that appears at 1658 cm-1 are attributed to the carbonyl amides present in the protein portion. The peak observed at 1587 cm-1 may be attributed to stretching connecting CN and also

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4000 3500 3000 2500 2000 1500 1000 500

Frequency (cm-1)

2852

Among the various techniques for material characterization, the X-ray diffraction (XRD) technique is recommended for the evaluation of the presence of crystalline phases present in natural materials. In general, we can classify materials as amorphous, semicrystalline or crystalline. Figure 8 shows the XRD patterns for *M. oleifera* seeds. The XRD pattern for crushed seeds, due to the high amount of oils and proteins present in the composition of the material which represent around 69% of the total mass [36], shows unresolved signals (predominantly amorphous). For this reason intact seeds are analyzed, constituting a complex matrix com‐ prisedofawidevariationofsubstancesincludingproteins,lipidstructuresand,toalesserextent, carbohydrates. It was possible to separate a broad peak at around 2θ equals 10º. The presence of this peak is probably associated with the diffraction of the protein constituent surrounded by other components which have a more amorphous pattern [37]. The amorphous nature of the biosorbent suggests that the metal ion could more easily penetrate the biosorbent surface.

Thermogravimetric (TG) analysis was used to characterize the decomposition stages and thermal stability determined through the mass loss of a substance subjected to a constant heating rate for a specified time. The mass loss curve for a sample of *Moringa oleifera* seeds can be observed in Figure 9, showing a typical profile that indicates several stages of the decom‐ position process. This thermogravimetric curve verifies the sample heterogeneity, since the intermediates formed are a mixture of several components. The mass loss curve can be divided into three stages: i) the first step occurs from 30°C to 128°C where a mass loss in the order of 8%, associated with water desorption, was observed. The amount of water loss from seeds determined by this technique is similar to the value of 8.9% found in [38]; ii) in the second step

3420 2923

**Figure 7.** FT-IR spectrum of *Moringa oleifera* seeds. The arrows indicate the maximum signal obtained [36].

1740

1800

1587

1658

1600

1715

the deformation of the N-H bond present in the proteins of seeds [34, 35].

Transmitance (%)

**Figure 6.** Schematic diagram of an ion exchange mechanism [29].

The seeds of *Moringa oleifera* and its parts can be classified as lignocellulosic adsorbents, consisting mainly of cellulose, hemicellulose and lignin. These functional groups are com‐ prised of macromolecules that have the ability to absorb metal ions through ion exchange or complexation [30] phenomena which occur on the surface of the material through the inter‐ action of the metal with the functional groups present. In order to understand the adsorption process it is also important to characterize the biomass material. Several techniques can be used to define the functional groups responsible for the adsorption phenomenon.

Infrared spectroscopy is an important technique in the qualitative analysis of organic com‐ pounds, widely used in the areas of natural products, organic synthesis and transformations. It is applied as a tool to elucidate the functional groups which may be present in substances [31], particularly with respect to the availability of the main groups involved in adsorption phenomena.

Figure 7 shows FT-IR spectra for *Moringa oleifera* seeds which verify the presence of many functional groups, indicating the complex nature of this material. The bandwidth centered at 3420 cm-1 may be attributed to the stretching of OH bonds present in proteins, fatty acids, carbohydrates and lignin units [32]. Due to the high content of protein present in the seed there is also a contribution in this region from N-H stretching of the amide bond. The peaks present at 2923 cm-1 and 2852 cm-1, respectively, correspond to asymmetric and symmetric stretching ofthe C-H bondofthe CH2 group.Due to the high intensity ofthese bands itis possible to assign them to the predominantly lipid component of the seed, which is present in a high proportion similar to that of protein [33]. In the region of 1800-1500 cm-1 a number of overlapping bands are observed and between 1750 and 1630 cm-1 this can be attributed to C=O stretching. Due to the heterogeneous nature of the seed, the carbonyl group may be bonded to different neighbor‐ hoods aspart ofthe fatty acids ofthe lipidportionor amides oftheproteinportion.The carbonyl component that appears due to the presence of lipids can be seen at 1740 and 1715 cm-1, as can be observed in the infrared spectra as small peaks, and the shoulders forming part of the main band that appears at 1658 cm-1 are attributed to the carbonyl amides present in the protein portion. The peak observed at 1587 cm-1 may be attributed to stretching connecting CN and also the deformation of the N-H bond present in the proteins of seeds [34, 35].

In reference [28] indicated that ion-exchange is an important concept in biosorption, because it explains many of the observations made during heavy metal uptake experiments. In this context, the term ion-exchange does not explicitly identify the mechanism of heavy metal binding to biomass, and electrostatic or London–van der Waals forces should be considered as the precise mechanism of chemical binding, i.e., ionic and covalent bonds. Figure 6 provides a schematic representation of an ion-exchange mechanism for a biosorbent material where

The seeds of *Moringa oleifera* and its parts can be classified as lignocellulosic adsorbents, consisting mainly of cellulose, hemicellulose and lignin. These functional groups are com‐ prised of macromolecules that have the ability to absorb metal ions through ion exchange or complexation [30] phenomena which occur on the surface of the material through the inter‐ action of the metal with the functional groups present. In order to understand the adsorption process it is also important to characterize the biomass material. Several techniques can be

Infrared spectroscopy is an important technique in the qualitative analysis of organic com‐ pounds, widely used in the areas of natural products, organic synthesis and transformations. It is applied as a tool to elucidate the functional groups which may be present in substances [31], particularly with respect to the availability of the main groups involved in adsorption

Figure 7 shows FT-IR spectra for *Moringa oleifera* seeds which verify the presence of many functional groups, indicating the complex nature of this material. The bandwidth centered at 3420 cm-1 may be attributed to the stretching of OH bonds present in proteins, fatty acids, carbohydrates and lignin units [32]. Due to the high content of protein present in the seed there is also a contribution in this region from N-H stretching of the amide bond. The peaks present at 2923 cm-1 and 2852 cm-1, respectively, correspond to asymmetric and symmetric stretching ofthe C-H bondofthe CH2 group.Due to the high intensity ofthese bands itis possible to assign them to the predominantly lipid component of the seed, which is present in a high proportion similar to that of protein [33]. In the region of 1800-1500 cm-1 a number of overlapping bands are observed and between 1750 and 1630 cm-1 this can be attributed to C=O stretching. Due to the heterogeneous nature of the seed, the carbonyl group may be bonded to different neighbor‐ hoods aspart ofthe fatty acids ofthe lipidportionor amides oftheproteinportion.The carbonyl

used to define the functional groups responsible for the adsorption phenomenon.

"Me" represents a metal with valence +2.

232 Applied Bioremediation - Active and Passive Approaches

**Figure 6.** Schematic diagram of an ion exchange mechanism [29].

phenomena.

**Figure 7.** FT-IR spectrum of *Moringa oleifera* seeds. The arrows indicate the maximum signal obtained [36].

Among the various techniques for material characterization, the X-ray diffraction (XRD) technique is recommended for the evaluation of the presence of crystalline phases present in natural materials. In general, we can classify materials as amorphous, semicrystalline or crystalline. Figure 8 shows the XRD patterns for *M. oleifera* seeds. The XRD pattern for crushed seeds, due to the high amount of oils and proteins present in the composition of the material which represent around 69% of the total mass [36], shows unresolved signals (predominantly amorphous). For this reason intact seeds are analyzed, constituting a complex matrix com‐ prisedofawidevariationofsubstancesincludingproteins,lipidstructuresand,toalesserextent, carbohydrates. It was possible to separate a broad peak at around 2θ equals 10º. The presence of this peak is probably associated with the diffraction of the protein constituent surrounded by other components which have a more amorphous pattern [37]. The amorphous nature of the biosorbent suggests that the metal ion could more easily penetrate the biosorbent surface.

Thermogravimetric (TG) analysis was used to characterize the decomposition stages and thermal stability determined through the mass loss of a substance subjected to a constant heating rate for a specified time. The mass loss curve for a sample of *Moringa oleifera* seeds can be observed in Figure 9, showing a typical profile that indicates several stages of the decom‐ position process. This thermogravimetric curve verifies the sample heterogeneity, since the intermediates formed are a mixture of several components. The mass loss curve can be divided into three stages: i) the first step occurs from 30°C to 128°C where a mass loss in the order of 8%, associated with water desorption, was observed. The amount of water loss from seeds determined by this technique is similar to the value of 8.9% found in [38]; ii) in the second step 32% of mass loss was observed in the temperature range of 128–268°C. This stage occurs due to the decomposition of organic matter, probably the protein component, present in seeds; and iii) the third step occurs from 268°C to 541°C with decomposition of the greater part of the seed components, which probably includes fatty acids, for example, oleic acid has a boiling point of 360°C. At 950°C a total residue of around 14.6% was observed, due to the ash content and probably inorganic oxides.

some deformations on the surface of the plant tissue can be observed, containing available sites, from which it is possible to infer that the adsorbent provides favorable conditions for the

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adsorption of metal species in the interstices [35].

(a) (b)

**4. Influence of parameters in biosorption process**

tion experiments are carried out in batch mode.

**Figure 10.** Scanning electron micrographs of *Moringa oleifera.* In the order of (a) 10 µm and (b) 50 µm [36].

Many variables can influence metal biosorption and experimental parameters such as tem‐ perature, stirring time, pH, particle size of the biomass, ionic strength and competition between metal ions can have a significant effect on metal binding to biomass. The biomass mass also influences the adsorption process because as the adsorbent dose increases the number of adsorbent particles also increases and there is greater availability of sites for adsorption. Some of the most important factors affecting metal binding are discussed below. In general, adsorp‐

The pH is one of the most important parameters affecting any adsorption process. This dependence is closely related to the acid-base properties of various functional groups on the adsorbent surfaces [39]. The literature shows that a heterogeneous aqueous mixture of *M. oleifera* seeds contains various functional groups, mainly amino and acids groups. These groups have the ability to interact with metal ions, depending on the pH. An increase in metal adsorption with increasing pH values can be explained on the basis of competition between the proton and metal ions for the same functional groups, and a decrease in the positive surface charge, which results in a higher electrostatic attraction between the biosorbent surface and the metal [40]. Low pH conditions allow hydrogen and hydronium ions to compete with metal binding sites on the biomass, leading to poor uptake. Biosorbent materials primarily contain weak acidic and basic functional groups. It follows from the theory of acid–base equilibrium that, in the pH range of 2.5–5, the binding of heavy metal cations is determined primarily by the dissociation state of the weak acidic groups. Carboxyl groups (–COOH) are important groups for metal uptake by biological materials. At higher solution pH, the solubility of a metal complex decreases sufficiently for its precipitation, leading to a reduced sorption capacity.

**Figure 8.** X–ray diffractogram for *Moringa oleifera* seeds [36].

**Figure 9.** Thermogravimetric curve for *Moringa oleifera* seeds [36].

The morphological characteristics of the crushed seeds obtained using a scanning electron microscopy (SEM) can be seen in Figure 10. The results reveal that the material exhibits a relatively porous matrix with heterogeneous pore distribution. This feature is attributed to the fact that the whole seed comprises a wide variety of biomass components. The presence of some deformations on the surface of the plant tissue can be observed, containing available sites, from which it is possible to infer that the adsorbent provides favorable conditions for the adsorption of metal species in the interstices [35].

**Figure 10.** Scanning electron micrographs of *Moringa oleifera.* In the order of (a) 10 µm and (b) 50 µm [36].
