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In this article, we focus on reviewing the recent progress in the field of citrus biomass research focused on producing energy, biofuels, important chemicals and bio-sorbent materials for the adsorption of dyes, heavy metals and toxic chemicals by processing of citrus waste biomass via biotransformation. Biotransformation is the modification of waste biomass or harmful chemical substances by certain microorganisms or chemical agents to render them either harmless to humans and the environment or for synthesis of useful products for safe consumption. The review has been presented in two parts under the titles "*Biotransformation of Citrus Waste-I: Production of Biofuel and Valuable Compounds by Fermentation*", and "*Biotransformation of Citrus Waste-II: Bio-sorbent Materials for the Removal of Dyes, Heavy Metals and Toxic Chemicals from Polluted Water*". The first part deals with the synthesis and production of biofuels (ethanol, methane and biodiesel) and valuable compounds, such as organic acids (citric, succinic, pyruvic, lactic, acetic), Vit-C, enzymes, single-cell proteins and prebiotics from fermentation of the citrus wastes [8]. In the second part of the article (present article), we attempt to review current and conventional trends of citrus waste disposal and their relative merits, and recent progress in biotransformation of citrus waste biomass into bio-sorbent material, employing physical, chemical or thermochemical methods for the adsorption of various pollutants, mainly heavy metals and dyes from polluted wastewaters and industrial effluents, and mechanisms and theoretical studies explored in the adsorption processes. The motivation behind this review is to conduct a detailed overview of the recent updates in this area of research and emphasize our focus towards the possibilities of harnessing the hidden potential of obtaining efficient products out of citrus biowaste, which otherwise is discarded in the dumping grounds as waste.

### **2. Methods of Preparation of Bio-Sorbents**

Bio-sorbents from citrus wastes have been developed in a number of ways, such as (a) mechanical shredding/grinding, (b) physicochemical treatment, (c) thermochemical treatment and (d) biochemical methods using enzymes. Based upon the treatment, the different kinds of bio-sorbents obtained from citrus waste can be classified into the following categories: (i) native peel bio-sorbent, (ii) protonated peel bio-sorbent, (iii) peel pectic acid bio-sorbent, (iv) de-pectinated peel bio-sorbent, (v) carbonized peel-activated carbon bio-sorbent, (vi) chemically modified bio-sorbent and (vii) biochemically or enzymatically modified bio-sorbent. The native peel bio-sorbents are obtained from physical or mechanical treatment of citrus peel biowaste. The biomass is washed with tap water to remove dirt, followed by washing with distilled or deionized (DI) water, or nano-pure (NP) water, 3–5 times. Washing is a common step and is essentially included in all the techniques of biomaterial preparation. The native peel bio-sorbent is developed from drying the washed peel waste at 323 K, until it attains a constant weight, followed by grinding and sieving to a desired particle size of ~0.5–1.0 mm, appropriate for adsorption studies. Peel pectic acid is prepared from albedo of the citrus peel waste. The peels are treated with hot acidified water (HCl, pH 1.5) at 358 K and stirred for 2 h. The treatment at elevated temperature facilitates extraction of pectic acid from the peel tissues. The extracted pectic acid is then filtered and coagulated with an equal volume of ethanol (95%). The coagulate is washed with ethanol and dried until constant weight. The peel pectic acid is insoluble in aqueous medium and remains stable during the adsorption tests [40]. The de-pectinated peel adsorbents are obtained from the residue remaining after the extraction of pectin from peel waste. The pectin is mainly extracted from the albedo (white) part of the peels. The residue is washed thoroughly and dried in an oven until constant weight [1,2].

Chemical modification of bio-sorbents is carried out in order to enhance the adsorption capacity by introducing active functional groups by means of chemical reactions. Furthermore, it helps in enhancing the chemical stability and mechanical properties of the sorbent material. The latter prohibits the release of pollutants into the adsorption system. Chemical treatment is carried out via several methods. Some of the well-known methods are: (a) protonation (inorganic acid: HCl, H2SO4, HNO3), (b) alkali saponification (using NaOH, Ca(OH)2, CaCl2), (c) phosphorylation, (d) blocking of functional groups

using chemical reagents, (e) organic acids (citric acid, oxalic acid), (f) H2O2 treatment, etc. Protonated peel bio-sorbents are obtained from protonation of the citrus peel waste collected after washing and drying. The protonation is carried out by treating the material with acids, such as HCl or HNO3. For this, the material is suspended in the acid and stirred or shaken for 6–8 h, followed by filtration, washing until neutral pH and drying until constant weight. Protonation is employed to remove excess of cations, such as Ca2+ or Na+, present on the biomaterial surface, which interfere with the metal sorption process. These cations are replaced by protons, which enhances the binding of heavy metals by decreasing the competition between Na+, K+ and Ca2+ ions with heavy metals such as Cd2+, Hg2+, Pb2+, Zn2+, Se2+, As2+ and so on. Replacement of Ca2+ and Na+ ions by protons has exhibited enhancements in the adsorption of desired heavy metal ions and their removal from wastewater. Treatment with NaOH and citric acid is employed to introduce carboxyl groups on the bio-sorbent surface, which interact with heavy metals to form a complex, and the resultant structure helps in the removal of the toxic elements from the wastewater. Phosphorylation is employed to introduce abundant alcoholic "-OH" groups and phosphoric groups into the bio-sorbent material. The latter, possessing a high affinity for ferric iron, enhances the loading capacity for iron. Iqbal et al. carried out experiments to block "-COOH" and "-OH" functional groups using anhydrous CH3OH, and concentrated HCl and HCHO, respectively. The modified bio-sorbents with blocked functional groups were found to exhibit a reduced adsorption capacity of Ni2+ by 78.57% and Zn2+ by 73.31%, confirming the main contribution of carboxyl and hydroxyl functional groups in the adsorption of heavy metal ions [69].

In the carbonization process, the dried peels are subjected to a very high temperature of ~773 K, followed by acid oxidation. An inert atmosphere, such as N2, is employed to prohibit fire or rigorous oxidation. The positively charged amine groups present on the surface of the adsorbent material facilitate binding to anionic RMB reactive dye by electrostatic attraction [70]. Bhatnagar et al. prepared a bio-sorbent from lemon peels by thermal activation at 323 K in the presence of air, which converted the peels to ash. Treating bio-sorbents with H2O2 has been employed to avoid the release of color in bio-sorbents [71]. Treating with 1% NaOH and ethanol removes lignin and colored pigments. Carbonization through the chemical activation method is one of the most favored methods for preparation of adsorbent material from citrus fruit peel. Weight ratios of peel vs. activating agent, temperature and time of carbonization are the selected parameters for optimizing the preparation of an efficient adsorbent material [72]. Generally, the dried citrus fruit peel is fed to a mixer grinder, and the ground powder is mixed with activating agents such as ortho-phosphoric acid, zinc chloride or sulfuric acid. This is then carbonized in a muffle furnace by heating it at an elevated temperature of ~723–823 K, up to a duration of 0.75 to 1.5 h. The weight ratio of dried citrus fruit peel to activating agen<sup>t</sup> varies in the range of 1:1 to 3:1. The charred material is then cooled and washed with dilute ammonia solution and distilled water. This removes any unconverted activating agen<sup>t</sup> from the carbonaceous material. The washing of the sample is continued until the pH becomes neutral. The charred material is then left for drying overnight under ambient conditions. The dried samples are then crushed and fractioned into different sizes [72].

Cross-linked hydrogel adsorbents for the removal of dye molecules can be prepared by treating the ground peel powder with N-vinyl-2 pyrrolidone (NVP), followed by irradiation with gamma rays. Mahmoud et al. demonstrated that a gamma irradiation dose of 30 kGy to the hydrogel precursor composition of NVP and orange peels in the ratio of 1:1 results in optional homogeneity of the bio-sorbent material with appropriate properties for practical applications. Usage of hydrogel (cross-linked polymerized hydrogel) enables the material to adsorb and retain large volumes of water and facilitate in increasing the contact time between the pollutant dyes and adsorbent material by the virtue of its cross-linked threedimensional network structure. A porous material possesses additional benefits in terms of extended surface area for adsorption [73]. Chemical treatment by formaldehyde and urea carried out by Rabia et al. demonstrated an enhancement in roughness and unevenness,

with apparent pores and canals of irregular shapes in a 3D network structure of the material surface to facilitate extended contact time between the adsorbate molecules and adsorbent material, and enhance physisorption [74]. Treating with HCHO improves the shelf-life of the bio-sorbent and prevents microbial damage [75]. While the functional groups, such as carboxylic acid, amine and sulfonic acid groups participate in electrostatic interaction and binding with dye molecules, the bulky structure of the hydrogel can be conveniently collected, separated and regenerated either by washing with water or treating with acid for further usage. Dev et al. reported on the adsorption of selenium from wastewater by citrus peel-based bio-sorbents chemically modified by calcium alginate. The chemical modification provides structural stability to the adsorbent material, which allows its reusability. Besides, it enhances the number of "-COOH" and "-OH" functional groups due to alginate beads, which enables the bio-sorbent material to absorb and remove other metallic ions in addition to SeO4<sup>2</sup>− and SeO3<sup>2</sup>− [76].

Kam and Lee carried out adsorption of amoxicillin onto the activated carbon surface prepared from citrus peel waste from aqueous solution containing the antibiotic, and reported an efficient adsorption within 30 min and attainment of equilibrium in 90 min. The waste citrus peel-based activated carbon showed a maximum adsorption capacity of 125 mg/g of the adsorbent at 293 K [77]. Putra et al. studied adsorption and removal of amoxicillin using commercial activated carbon [78]. Moussavi et al. reported on the adsorption studies of amoxicillin on commercial activated carbon and activated carbon derived from pomegranate wood [79]. Ding et al. developed activated carbon from sewage sludge and oil sludge and reported adsorption and removal of oxytetracycline and chlortetracycline [80]. Baccar et al. obtained activated carbon from olive-waste cake to absorb naproxen, ketoprofen, diclofenac and ibuprofen from aqueous solution containing the contaminants [81]. Ahmed et al. produced activated carbon from Siris seed pods and carried out adsorption and removal of metronidazole from contaminated water [82]. On the other hand, activated carbon developed from vine wood by Pouretedal and Sedech et al. showed efficient adsorption of amoxicillin, cephalexin, tetracycline and penicillin from contaminated water [83].

The adsorption of heavy metal ions onto the adsorbent surface is influenced by several factors, such as nature of the material, charge on the chelating metal ion, size of the ion, nature of donor atom present in the ligand, buffering environment during the adsorption process and exchange of ions, nature and properties of the solid support, and so on [84]. In this direction, Li et al. carried out chemical modification employing 20% isopropyl alcohol to remove coloring compounds from orange peels, along with polar compounds. This was followed by saponification by addition of 0.1 M NaOH/0.1 M NH4OH/saturated solution of Ca(OH)2. The Na+/NH4+ or Ca2+ ions become attached to the cellulose molecules of the adsorbent material and facilitate an ion exchange mechanism between Na+, NH4+ or Ca2+ ions and the bivalent heavy metal ions. In the next step, the saponified orange peels are treated with 0.6 M acid at an elevated temperature of 353 K. The heat is required to produce a condensation product and acid anhydride. The latter combines with cellulose hydroxyl groups and results in the formation of ester linkage and introduction of carbonyl groups to the cellulose molecule. The additional carbonyl functional groups introduced to the cellulose molecule enhance metal ion adsorption [85]. In another method, Liang et al. treated orange peels with NaOH followed by mercaptoacetic acid (C2H4O2S) in order to convert the hydroxyl groups present in the cellulose molecules into mercapto groups. The latter exhibited a higher affinity towards heavy metal adsorption (Cu2+ and Cd2+) from aqueous solutions [86]. The final different sized samples are used for adsorption purposes. The different methods of bio-sorbent pre-treatment by heat, chemical(s) and enzymes are summarized in Figure 6.

**Figure 6.** *Cont.*

**Figure 6.** (**<sup>a</sup>**–**<sup>t</sup>**) Different methods of pre-treatment of the precursor material for the preparation of bio-sorbents from citrus wastes [69–76,84–88].

Cameron et al. carried out an elaborated study on the adsorption of Pb2+ ions by adsorbents synthesized from citrus peels and peel-derived pectin and concluded that fragmentation of larger molecules into smaller fragments and demethylation of the same occur. The latter plays an important role in the enhancement of the sorption capacity of pectin and derived materials. The fragmentation can be carried out either chemically or enzymatically. Pectin, a polysaccharide present in the citrus peels, can be modified via enzymatic or chemical conversion to develop a suitable bio-sorbent as well as fine-tune the desired properties, e.g., ion exchange and adsorption properties. Pectin polymer is made of galacturonic acid (GA) monomer units, a major sugar found in citrus fruits. It is basically concentrated in the linear homogalacturonan region (HG), which is pectin's dominant structural domain. The carboxylic acid functional groups present in the GA molecular structure interact with the heavy metal cation present in the wastewater or industrial effluents and require removal. While the polyanionic character of pectin is crucial for adsorption, masking of the negative charge present on the carbonyl group in the GA molecule by means of methyl esterification at C-6 position hence alters the overall functionality of the pectin molecule. In other words, the pectin functionality is dependent upon the total amount of methylation of GA units or degree of methylation (DM) and distribution of methylated GAs and non-methylated GAs (GAs with unmasked or free carboxylic acid functional groups) in the pectin polymeric chain in the HG region [89]. The de-esterification of the GAs or de-methylation of GAs can induce ordered or random distribution of de-methylated GAs in the pectin polymer chain. Both kinds of specific properties obtained post-modification, e.g., degree of methylation (DM) and degree of polymerization (DP), in pectin molecule have been reported to exhibit effects in terms of interaction with cation and sorption properties [87,88]. Pectin extraction can be carried out via aqueous extraction using an aqueous acid or base followed by purification of liquid extracts containing hydrocolloids, and isolating the extracted pectin from the mixture [1,2]. There is a basic difference in the product quality of pectin obtained post-acid/alkali treatment. The pectin obtained from the alkali extraction process contains a low degree of esterification (low DE pectin). The latter results from saponification of the ester groups present in the polymer molecule by alkali. On the other hand, pectin obtained from the acid extraction process contains a high degree of esterification (with high DE pectin ~50% and greater) [89]. The methods of obtaining bio-sorbent materials from citrus peel-derived oligosaccharides and enzymatically modified pectin have been summarized in Figure 7a, and their respective adsorption capacities towards Pb2+ are shown in Figure 7b.



**Figure 7.** (**a**) Preparation of bio-sorbent from biochemical treatment of citrus peel waste. (**b**) Bio-sorption capacities of different bio-sorbent substrates prepared in (**a**). Adapted from the information provided in References [89–91].

### **3. Adsorption Experiments and Mechanism**

The bio-sorption of harmful chemicals from wastewaters has been focused on achieving two prime targets: (a) development of novel bio-sorbent substrate material, and optimization of the adsorption process in terms of uptake of pollutant molecules/ions in a batch reactor containing a single target metal, and (b) enhancement of sorption uptake capacity by suitable processing [92,93]. From the viewpoint of carrying out adsorption processes/experiments, either or both of the two main techniques are employed, namely, batch adsorption test and/or column adsorption test. In the batch adsorption process, ion exchange has been identified as the main mechanism for the adsorption and removal of heavy metal ions or dye molecules. The carboxyl and hydroxyl functional groups present on the adsorbent substrate have been demonstrated to play key roles in the adsorption process. Other functional groups, such as amide, sulfonate, phosphate and amino groups, have also been reported to participate in the adsorption process. The adsorption process is a complex interplay between a number of mechanisms, such as complexation, coordination, chelation, ion adsorption or exchange, micro-precipitation, electrostatic interaction, H-bonding and so on. In the experimental part, a fixed amount of bio-sorbent substrate/material is placed with a definite volume of solution containing the pollutant or toxic metal ions/dyes, and stirred/shaken for a specified duration in a beaker or flask. After the adsorption process is completed, the solution is filtered and the adsorbent is regenerated or recycled by washing with water or treating with chemicals, such as acids, alkali or organic solvent(s). Alternately, the bio-sorbent material can also be regenerated by physical or thermo-physical treatments, such as heating, microwaving or sonication [93–95].

In some cases, the test solution has been observed to develop a brown color, which is explained by leaching out of carotenoids from the citrus peel biomass. Carotenoids are responsible for binding the heavy metal ions present in the test solution and forming complexes. The latter has been found to be unable to adsorb onto the bio-sorbent surface, and hence, left in the test solution imparting color. This problem can be solved by chemical pre-treatment of the bio-sorbent material, which helps in leaching out the carotenoid or other colored materials, such as chlorophyll, from the citrus peel biomass. Treating the biomass with isopropanol helps in the removal of soluble compounds without any adverse effect on the nature of biomass material or ion-binding sites on the surface. Citric acid treatment helps in the dissolution of polysaccharides present in the cell wall of the biomass. This facilitates in opening up the physical structure of the biomass and thereby increases the number of adsorption/binding sites, i.e., functional groups. Alkali treatment has been observed to impart a stronger effect on cell wall rupturing and facilitate the exposure of functional groups. Furthermore, the hydroxyl or carboxylic acid groups are converted to their salt forms, thereby helping to enhance the adsorption process by an ion exchange mechanism. An increase in temperature during alkali or acid pre-treatment has been observed in biomass loss. The factors influencing the bio-sorption process are initial pH of the test solution, concentration of the test solution (heavy metal ions, dye molecules, etc.), dosage of adsorbent, pre-treatment of the bio-sorbent, temperature during the adsorption process and duration of the contact time between the sorbent material and the test solution. The advantages of the batch adsorption technique are short analysis time, low operational costs, simple maintenance and conductance and that it can be operated with locally available adsorbent materials with satisfactory efficiency [96].

A next-level technique, which can be employed for large sample sizes or industrial scale purposes regarding adsorption and removal of pollutants, is fixed-bed reactors or the column adsorption method. It is also called a continuous flow column system. The fixedbed column is a commercially viable technique, and at present, employed in industries fitted with ion-exchange resins or other commercially available adsorbent materials. The column material is usually made of clear-extruded acrylic. The length of the column ranges between 25 and 30 cm with an internal diameter of 1.3–1.5 cm. The bottom layer of the column is filled with spherical glass beads (of diameter 3 mm) to facilitate even distribution of influent flowing across the column length. A fiber screen is placed at the top of the

column to prohibit the scattering of the adsorbent material in the fixed-bed and confine it appropriately in a fixed position during the entire operation. The wet-packing of the column is usually performed in such a way so as to allow a void space inside the column up to 70%. The bed-height is hence adjusted up to 24 cm in order to enhance the length of the fixed-bed, and the column length can be increased by supplementing an additional number of columns in series. During operation, a certain amount of material by weight is wetpacked in the column and the column is conditioned with a suitable solvent (e.g., acidified water of pH 4.0–5.5) overnight. Post-conditioning, the test solution (prepared at the same pH) is percolated through the column at a constant flow rate using a peristaltic pump or a micro-tube pump. The test solution passes through the column contents, i.e., packed adsorption bed, and the latter adsorbs the heavy metal ions or dye molecules, leaving behind the solution with a lesser or minimal concentration of pollutants. If the driving force (concentration variation between sorbent and influent) remains high, the sorbent shows high metal ion uptake. This is due to the adsorbent material being saturated at a relatively high influent concentration, whereas the progressing metal solution comes repeatedly in contact with a fresher and more efficiently adsorbing surface, and thus the effluent leaving the column becomes virtually free of metal ions. The saturated column bed can be regenerated and recycled. Regeneration of the column bed is carried out by eluting the bed with a suitable desorption solution. The effluent samples are collected at a fixed interval of time using a fraction collector [97,98]. The schematic representation of the experimental set-up for the fixed-bed adsorption column is shown in Figure 8. The important reported results of experiments on bio-sorbent materials developed from citrus peel waste are listed in Tables 2–4. The various mechanisms of adsorption of heavy metals and dyes on the biomass derived bio-sorbent surface are shown in Figure 9.

**Figure 8.** Schematic representation of experimental set-up for fixed-bed adsorption column. Artwork developed from References [97–99].


**Table 2.** Citrus waste-based bio-sorbents for removal of heavy metals from wastewater (BAT: Batch Adsorption Test; DAT: Direct Adsorption Test (Column Adsorption)).

**Table 3.** Citrus waste-derived bio-sorbents via heat/chemical and enzyme pre-treatment for removal of heavy metals from wastewater.







**Table 4.** Citrus waste reuse as bio-sorbents for the removal of poisonous dyes from wastewater.





**Figure 9.** Mechanism of adsorption of dyes and heavy metals from industrial wastewaters by citrus biomass-derived bio-sorbent. Artwork developed from the information provided in [47].

### **4. Kinetics and Thermodynamics**
