**Thermochemical Route for Extraction and Recycling of Critical, Strategic and High Value Elements from By-Products and End-of-Life Materials, Part I: Treatment of a Copper By-Product in Air Atmosphere**

#### **Ndue Kanari 1,\*, Eric Allain 1, Seit Shallari 2, Frederic Diot 1, Sebastien Diliberto 3, Fabrice Patisson 3 and Jacques Yvon 1**

Received: 26 April 2019; Accepted: 15 May 2019; Published: 17 May 2019

**Abstract:** Development of our modern society requests a number of critical and strategic elements (platinum group metals, In, Ga, Ge ... ) and high value added elements (Au, Ag, Se, Te, Ni ... ) which are often concentrated in by-products during the extraction of base metals (Cu, Pb, Zn ... ). Further, recycling of end-of-life materials employed in high technology, renewable energy and transport by conventional extractive processes also leads to the concentration of such chemical elements and their compounds in metallurgical by-products and/or co-products. One of these materials, copper anode slime (CAS), derived from a copper electrolytic refining factory, was used for this study. The sample was subjected to isothermal treatment from 225 to 770 ◦C under air atmosphere and the reaction products were systematically analyzed by scanning electron microscopy through energy dispersive spectroscopy (SEM-EDS) and X-ray diffraction (XRD) to investigate the thermal behavior of the treated sample. The main components of the anode slime (CuAgSe, Cu2-xSeyS1-y, Ag3AuSe2) react with oxygen, producing mostly copper and selenium oxides as well as Ag-Au alloys as final products at temperatures higher than 500 ◦C. Selenium dioxide (SeO2) is volatilized and recovered in pure state by cooling the gaseous phase, whilst copper(II) oxide, silver, gold and tellurium remain in the treatment residue.

**Keywords:** critical and strategic materials; valuable metals; by-products; copper anode slime; thermal treatment; selenium dioxide

#### **1. Introduction**

Nowadays, besides the most common metals (Fe, Al, Cu, Zn, Ni ... ), a wide range of chemical elements (metals, non-metals, and metalloids) are in increasing demand and crucial for the development of renewable energies, the manufacture of electrical and electronic equipment, transportation, advanced industries, which constitute the foundation of innovative high technologies. Often suffering from a slight subjective and arbitrary classification, these elements known as rare elements, or sometimes as small, green, or high-tech metals, are commonly considered to be critical and strategic materials. According to criticality assessments, the number of critical materials (CRMs) for the European Union

has grown from 14 CRMs in 2011 to 20 in 2014 and was augmented thereafter to reach 27 CRMs in 2017 [1].

While the importance of CRMs in our society and their inventory is clear, the fragility of their supply represents a real challenge for the French, European and global economies. In general, material savings, substitution and recycling (including eco-design) can mitigate the risk of anticipated shortage of critical metals. Critical and strategic materials are often used in unique applications with limited alternatives and substitutions [2]. Note that the actual and future demand for several of these materials are expected to strongly grow, especially for the production of electric and electronic devices, while the life-spans of many products such as computers and cell phone are decreasing rapidly [3]. This phenomenon, among others, leads to the generation of a huge waste stream (waste electrical and electronic equipment-WEEE), often designated as "e-waste" [4–7]. Its elevated contents in high added values elements (precious, strategic, critical, rare and rare earths elements) make this waste stream a very attractive "urban mine" for the extraction of these components.

But paradoxically, the rate of recycling of CRMs from end-of-life materials is low compared to that of base metals. The presence of several metals of different chemical nature in increasingly miniaturized products, and incorporated in complex matrixes encompassing ceramics, resins, plastics, and especially halogenated substances (based on chlorine and bromine) makes their recycling process quite complex. A spectrum of contributions focused on current state-of-the-art research on metal recycling from wastes was introduced in a special issue (Valuable Metal Recycling) of *Metals* [8]. An extensive overview for the recovery of high value metals from electronic waste and spent catalysts using improved pyrometallurgical and hydrometallurgical technologies was given by Ding et al. [9]. It was mentioned that smelting is beneficial to segregate and concentrate the precious metals from e-waste in the by-products. In a recent investigation, Avarmaa et al. [10] demonstrated that during smelting, copper acts as collector metal for gold, silver, palladium and platinum contained in the end-of-life electronic devices. One may emphasize that most of the targeted elements (Au, Ag, Se, Te, platinum group metals, etc.) are recovered in anode slime from primary copper extraction from its sulfide ores and concentrates. In summary, recycling of 'e-waste' by pyrometallurgical route in the existing copper metallurgy leads to the concentration of these elements in by-and/or co-products, mostly in the anode slime of copper electrorefining.

A number of research reports were devoted to extraction and recovery of specific elements from copper anode slime (CAS). The most typical investigations concerned CAS treatment by H2SO4-O2 to recover Cu followed by thiourea leaching for Ag extraction [11] or leaching in a sulfuric and nitric acid medium which was used for silver nanoparticles synthesis [12]. A nitric-sulfuric acid mixture was also tested by Li et al. [13] for the recovery of Cu, Ag and Se from a CAS sample, while Xiao et al. [14] reported an oxidative pretreatment by thiosulfate leaching followed by an electrodeposition process for the selective recovery of Ag. Dissolution of selenium in NaOH, after sulfuric acid decopperization of CAS, was also reported [15]. However, little recent information is available for the thermal behavior of CAS under an oxidizing atmosphere. In this frame, the work presented in this article deals with the treatment in an air atmosphere of a CAS sample generated by a European copper plant. Particular attention was payed to the speciation and physical characterization of the obtained products (residues and condensates) at various temperatures to determine the reaction mechanisms and to identify process steps for a possible selective separation of the CAS components.

This research work is an extension of our investigations on the concentration and extraction of a broad range of valuable metals from raw materials and solid industrial residues by thermal route [16–20] as well as on the recycling and valorization of co-products and wastes via chemical synthesis of new materials [21–24].

At least two of the researched elements of this investigation, selenium and tellurium, belong also to the scattered elements category; i.e., which exist at low content, in a decentralized state and rarely form independent minerals in nature [25]. Both elements are used in thin films (CIGS—copper indium gallium selenide and CdTe—cadmium telluride) and used in second-generation modules of photovoltaic panels [26–28]. Efficient extraction and recycling of these elements from by-products, end-of-life solar photovoltaics and other wasted materials will be a main research focus in the future to meet the volume of industrial demand.

#### **2. Materials and Methods**

A sample of copper anode slime provided from a refining factory available as a powdered solid with a mean particles size less than 30 μm was used for this study. The sample composition was determined by diverse analytical methods, but only the results of scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS) and X-ray diffraction (XRD) analyses were selected to be presented in this paper (see Section 3). SEM examinations were performed by using a HITACHI S-4800 device (Hitachi Ltd., Tokyo, Japan) equipped with an EDS elemental analysis microprobe. SEM imaging and chemical microanalysis were performed mostly at an accelerating voltage of 15 kV. The samples were carbon coated prior to analysis. XRD was performed on samples using a Bruker D8 Advance device (Bruker, Karlsruhe, Germany), (Co Kα1 radiation, λ = 1.789 Å), under 35 kV and 45 mA operating conditions. The acquisition of diffraction patterns was performed between 3◦ and 64◦ (2θ) range at step scan 0.034◦ with 3 s per step. The XRD-patterns were analyzed thanks to the DIFFRAC.EVA software (version 5, Bruker) and PDF-2 release 2011 database.

Isothermal experimental tests for the CAS sample treatment were conducted in horizontal set-ups, including a system of static tubular furnaces that were able to reach 1600 ◦C. To achieve this, a pre-weighted sample of several grams was introduced directly into the furnace preheated at the desired temperature. When the dwell time was reached, the sample was removed from the furnace and cooled down to room temperature. Air was used as a flowing gas and it also performed the oxidation of CAS components. The outlet gases were cooled at room temperature, leading to the condensation of the vapor phase and the recovery of a solid condensate. Initial CAS sample and solid products obtained from CAS thermal treatment were examined by visible microscopy, SEM-EDS and XRD. The advantage of using SEM-EDS analysis here is the ability to gather punctual information about elemental content, phases differentiation, and about the morphological and textural evolution of the thermally treated samples, all this contributes to a better understanding of the involved reaction mechanism and processing steps.

#### **3. Results**

#### *3.1. Elemental and Mineralogical Analysis of CAS Sample*

Several research reports [11–15,29–32] found in the literature revealed that the copper anode slime is a quite complex material in elemental and mineralogical composition, as well as in morphological aspects. This complexity resulted from the nature of copper raw materials, operating conditions of copper extractive processes (smelting, converting, fire- and electro-refining) and from the decopperization step applied to treat CAS. By the same token, a general SEM image of the used CAS sample for this study given in Figure 1a shows a particular morphology of this solid. Besides the spherical particles, there are many particles that are irregularly and shell shaped. As clearly shown by the SEM image, most particles are corroded, resulting in three-dimensional lens-like forms and other ones with plenty of cavities and pores.

The general EDS spectrum of the CAS (Figure 1b) indicated a multi-elemental composition with a descending peak intensity order as follows: Cu, Se, Ag, O, S, Au, Te, Si, Cl. There is probably also a carbonaceous matter in the CAS sample, but it was difficult to distinguish from the carbon coating used to make the sample conductive before SEM-EDS analysis.

**Figure 1.** SEM-EDS results of initial copper anode slime: (**a**) General view (backscattered electron micrograph) of the used sample; (**b**) Overall EDS analysis of the used sample.

The morphology of the CAS sample showed in Figure 2 and SEM-EDS microanalysis data summarized in Table 1, indicates that there are particles containing mostly Cu-Se (spot n◦ 1 and 2), Ag-Se-Cu-Te (spot n◦ 3) and Ag-Se-Cu-Au-Te (spot n◦ 4); gold is found mostly in the finest particles. There is also some sulfur in almost all particles, while the small amount of oxygen is bound to silicon. This peculiar state of CAS particles together with their composition can be explained by the preceding impact of at least two factors: (i) the behavior the Cu, Ag, Au, Se, Te, S, O and others elements compounds in fire refining process of blister copper and (ii) the chemical and electrochemical reactivity of these compounds towards electrolyte during electrorefining of anode copper. The fire refining process, with the goal of removing residual oxygen and sulfur as well as other impurities from blister copper before casting into anode, is achieved at 1200–1250 ◦C.

As could be expected, the residual oxygen and sulfur can be associated with copper forms as Cu2O and Cu2S, having higher melting points than copper. One may deduce that during the cooling of casted Cu-anodes, the Cu2O and Cu2S particles, which are first to be solidified, can serve as heterogeneous nuclei for further solidification around these nucleation sites. The phase diagrams for copper with chalcogen (e.g., S, Se, or Te) show that there are miscibility gaps in the liquid phase [33] that will allow the solidification of droplets with the highest melting points followed by further solidification of low melting point components, thereby resulting in a layered structure and in a significant difference between the core and the periphery of the obtained solid. During copper electrorefining, the copper oxide (CuO) contained in mixed (Cu, Se, Te, S, O ... ) particles as well as common impurities (most probably oxides of As, Sb and Bi) are dissolved, besides electrochemical dissolution of Cu◦, resulting in the final configuration of CAS. These observations are in good agreement with other research works reported in the literature [29–32].

**Figure 2.** Detailed aspects (backscattered electron micrograph) of initial CAS as revealed by SEM-EDS.

**Table 1.** Elemental composition of initial CAS analyzed by SEM-EDS.

1 wt % and at % represent mass and atomic percentage, respectively.

XRD results of the used CAS are shown in Figure 3. The main crystallized phase is Eucairite (CuAgSe), confirming the microanalysis obtained by SEM-EDS analysis. However, this phase probably forms during the electro-refining process. According to Chen and Dutrizac [29,32], silver is in a metastable state in the copper anode and this can promote the chemical and/or electrochemical dissolution of Ag◦ during electrorefining. The resulting ion Ag+ can react following Equation (1) by synthetizing the Eucairite phase. In addition, the authors stated that the particles kept the original morphologies of Cu2Se [Cu2(Se,Te)], although their composition has been changed. The conditions of the electrorefining process (mean values: T ≈ 60–65 ◦ C; ≈ 180–185 g/L H2SO4; residence time of several days) will certainly favor such a reaction.

$$\text{Ag}^+ + \text{Cu}\_2(\text{Se}, \text{Te}) \rightarrow \text{AgCu}(\text{Se}, \text{Te}) + \text{Cu}^+ \tag{1}$$

**Figure 3.** XRD patterns of CAS raw sample.

In the XRD patterns of CAS, there are some peaks corresponding to Cu2Se, Cu2S, CuSeS, as well as to non-stoichiometric ones with an absence of certain peaks due probably to crystallite orientation; for simplification, all of these phases are designated as copper selenide sulfide (Cu2−xSeyS1−y). Regarding the Au-bearing phase, the XRD peaks of the obtained diffractogram match most frequently with Fischesserite (Ag3AuSe2). This phase composition (with substitution of Ag by Cu and Se by Te) is also confirmed by SEM-EDS analysis (spot n◦ 3 and spot n◦ 4 in Table 1). Finally, Quartz (SiO2) completes the set of crystallized phases identified in the CAS sample.

#### *3.2. Thermal Treatment of CAS in Air for 1 h*

Several grams of the copper anode sample were used for isothermal tests performed between 225 ◦C and 770 ◦C in air atmosphere with a flow rate of 25 L/h. The evolution of the % mass loss (%ML) as a function of temperature is plotted in Figure 4. The %ML curve shape suggests that many reactions steps may occur in the explored temperature interval. The sample started to react with air oxygen (O2) at temperatures higher than 225 ◦C, resulting in a mass gain of up to about 350 ◦C. From here, there is a continued mass loss up to around 700 ◦C, where the mass loss is stabilized.

**Figure 4.** Evolution of the mass loss of the sample versus temperature during treatment of CAS in air for 1 h.

According to the composition of raw CAS sample, the main constituents which may strongly affect the %ML are Cu, Se and Ag combined as selenides of copper and silver. During their reactions with air (oxide formation) and knowing the atomic mass of these elements (Cu: 63.55; Se: 78.97 and Ag: 107.87, O: 16.00), it appears that no volatilization of these elements and/or their oxides occurred, at least up to 370 ◦C, since only a mass gain was recorded. The most volatile compound of the Cu-Se-Ag-O system is SeO2, having a vapor pressure of 73 kPa at 320 ◦C [34] which is enough for its volatilization during the CAS treatment at this temperature. This is an indirect confirmation that no free SeO2 is formed at T < 320 ◦C when CAS sample is heated in air. More consistent information about the interaction steps of CAS components with air is given in the subsequent section.

#### *3.3. Analysis of the Reaction Products*

The treatment residues issued from isothermal tests of CAS under air atmosphere were systematically examined by XRD technique and results are presented in Figures 5 and 6 for the treatment temperatures of 225–415 ◦C and 505–770 ◦C, respectively.

As shown in Figure 5, the X-ray diffractogram of the residue obtained at 225 ◦C is similar to that of the CAS raw sample. The XRD patterns of the sample heated 320 ◦C exhibits a fair broad diffraction band near to 6◦ (2θ), indicating a slight XRD disordered structure, at least, that of a reaction product which will be a precursor of further crystallized phases. Eucairite (CuAgSe) seems to be still stable at these temperatures, while the characteristic peaks of Cu2−xSeyS1−y and Ag3AuSe2 phases disappeared. Apparition of new crystallized phases such as: [Cu4O(SeO3)3], [Cu2O(SeO3)] and Ag◦ is obvious from the XRD examination. One may hypothesize that [Cu4O(SeO3)3], which can be written also as [4CuO·3SeO2], is the first crystallized phase issued from the reaction of copper selenide bearing substances with O2, though other phases not detected by XRD may be the intermediate product of selenide oxidation by oxygen. Metallic silver is also identified in this treatment residue; note that the main XRD peaks of Ag◦ and Au◦ overlapped. Further increase in the treatment temperature (from 320 ◦C to 415 ◦C) of CAS sample leads to a decrease in the reflection intensities of CuAgSe and Cu4O(SeO3)3 in the generated residue, while the relative intensity of [Cu2O(SeO3)] peaks seems increased. As noted by Portnichenko et al. [35], copper(II)-oxoselenite [Cu2OSeO3] has a cubic structure with a cell parameter a = 8.925 Å.

**Figure 5.** XRD patterns of CAS raw sample and the residues obtained during CAS sample treatment under air, from 225 ◦C to 415 ◦C.

At 505 ◦C (Figure 6), [Cu2O(SeO3)] is still a main crystallized phase, but a new phase, tenorite (CuO) is also identified and becomes the predominant phase at 600 ◦C. Based on the %ML of Figure 4, on the results of XRD diffraction and data from the research work performed by Fokina et al. [36], the two steps of Cu4O(SeO3)3 conversion into CuO and SeO2 as final products can be described by Equations (2) and (3). Diffractograms of products obtained at 685 ◦C and 770 ◦C confirm the presence of well-crystallized phases of silver (gold), tenorite and quartz, which was present in all analyzed residues. Though some sulfur is present in the CAS sample, often substituted by selenium and/or tellurium, no solid phase containing sulfur was identified in the thermal treatment residue.

$$\text{Cu}\_4\text{O(SeO}\_3\text{)}\_3 \to 2\text{Cu}\_2\text{O(SeO}\_3\text{)} + \text{SeO}\_2 \tag{2}$$

$$2\text{Cu}\_2\text{O(SeO}\_3) \to 4\text{CuO} + 2\text{SeO}\_2 \tag{3}$$

One may emphasize that no complete and systematic data of XRD analysis were found in the literature for monitoring the behavior of such copper anode slime under air in a wide temperature interval (225–770 ◦C). A comparison of XRD patterns of a CAS raw sample and its reaction product resulting from the thermal treatment of CAS at 770 ◦C in air is given in Figure 7. In summary, with a complex phase composition of a raw CAS sample (Eucairite-CuAgSe; copper selenide sulphide-Cu2−xSeyS1−y; Fischesserite-Ag3AuSe2 and Quartz-SiO2), the treatment residue at 770 ◦C is free of selenium bearing compounds and it is composed of cupric oxide, quartz and silver (gold). These compounds are concentrated in the residue as the thermal treatment allowed the volatilization of selenium dioxide, leading to a mass loss of sample exceeding 25 percent.

**Figure 6.** XRD patterns of the residues obtained during CAS sample treatment under air from 505 ◦C to 770 ◦C.

Likewise, several residues were examined by SEM-EDS technique. A typical morphology is given in Figure 8 with a visible difference in contrast suggesting the presence of areas of different compositions. Data of EDX analysis of spots 1 through 4 are summarized in Table 2. Within the measurement errors, the particle n◦ 1 is essentially composed of CuO (tenorite). The EDS analysis of spot n◦ 2 showed that silver (c.a., 78.6 wt %) is the major constituent with some gold (c.a., 14.6 wt %) and copper (c.a., 4.6 wt %). A possible zonal fusion may explain the smoothed shape of this particle. Copper and tellurium oxide (TeO2) seem to be the main components of the area noted as n◦ 3. The composition of crystal-like n◦ 4 is similar of spot n◦ 2 with a quite different morphology. Note that selenium is not detected in the treatment residue. These data seem to confirm those obtained by XRD diffraction analysis.

**Figure 7.** XRD patterns of a CAS raw sample and its treatment product in air at 770 ◦C.

**Figure 8.** SEM aspects (backscattered electron micrograph) of the CAS sample treated at 770 ◦C in air.

**Table 2.** Elemental composition (EDS data) of the CAS treated at 770 ◦C in air atmosphere.

1 w% and at % represents mass and atomic percentage, respectively.

During the treatment of CAS at temperatures above 500 ◦C, a solid mass is condensed in the tepid inner side of the experimental reactor. The visually observed quantity of this solid increased with the reaction progress and the rising temperature. A visible microscopy image of the recovered condensate is given in Figure 9 where centimetric needle-like crystals are observed. The SEM-EDS examination (Figure 10) showed that the solid is homogenous, nevertheless some small zones of spherical and other shapes characterized by micronic sizes were revealed. As shown by Figure 10b, the matrix area of SEM image (Figure 10a) is composed especially of Se and O in atomic proportion close to the SeO2 stoichiometry. In contrast, the lighter areas (Spot n◦ 2 in micrograph of Figure 10a) have a high content of selenium. Reduction of SeO2 to Se◦ is probably achieved by sulfur dioxide (SO2) during cooling of the gas phase. Taking into account that the crystallized phases synthesized during the oxidation of CAS by air (Figures 5 and 6) were [Cu4O(SeO3)3] and [Cu2O(SeO3)], the selenium dioxide (SeO2) which is generated from the decomposition of these compounds according to Equations (2) and (3) can be separated by volatilization and recovered in solid state.

**Figure 9.** Visible microscopy image of the solid condensate obtained during CAS treatment at 685◦C.

**Figure 10.** SEM-EDS results of a condensate obtained during thermal treatment of the Cu-anode slime sample: (**a**) General view (backscattered electron micrograph) of SEM with two different phases; (**b**) EDS analysis of the same phases.

This study encompassed some characteristics for the thermal treatment of a copper by-product and allows understanding of the behavior of selected element compounds during the process. However, the end-of-life materials (e-wastes, thin films of used photovoltaics, spent catalysts, wasted flat screen ... ) containing rare, critical, strategic and high value elements present also a drawback, which is the presence of plastic materials. In addition, these plastics contain a considerable amount of halogenated substances based on chlorine (polyvinyl chloride-PVC) and bromine (Br-flame retardants combined with based Sb-synergist). The thermodynamic and kinetic reactivity of these substances with respect to targeted metals during an envisaged thermal process will be the subject of the second part of this investigation.

#### **4. Conclusions**

With grown in demand of current and future development of new technologies for rare, critical and strategic elements and facing the depletion of basic primary resources, the targeted element extraction and recycling from by-products, wastes and end-of-life materials is a promising choice. Thermal treatment under an oxidizing atmosphere up to 770 ◦C of the copper anode slime (CAS), a potential source of these elements, led us to reach the following conclusions:

Selenium is mostly found in the copper-silver bearing phase (CuAgSe) and/or bounded to copper. Tellurium and sulfur can substitute selenium in the phase formulation. Gold is found in finest particles and Ag3AuSe2 seems to be the most likely gold bearing phase.

The thermal treatment of CAS at low temperatures led to the formation of double Cu-Se oxide [Cu4O(SeO3)3] which decomposed at temperatures higher than 400 ◦C.

Copper(II)-oxoselenite [Cu2O(SeO3)], synthetized also at low temperatures, is stable, at least up to 530 ◦C. At higher temperatures, only tenorite (CuO) is found in the treatment residues as a decomposition product of these double oxides, while selenium dioxide is volatilized.

Almost pure SeO2 is recovered by condensation of outlet gases issued from thermal treatment of CAS at temperatures higher than 500 ◦C.

The final residue of the CAS treatment under air beyond 600 ◦C is mostly composed of CuO and alloys of Ag-Au (with some Cu◦), while tellurium is found as oxide (TeO2), and silica (SiO2) remains intact. Further treatment of such material is required for the final separation of these high value element compounds.

**Author Contributions:** Conceptualization, N.K., E.A., F.P. and J.Y.; Formal analysis, S.S., S.D. and F.D.; Investigation, N.K., S.D. and F.D.; Visualization, S.S. and F.D.; Resources, N.K., F.P. and J.Y.; Writing—original draft, N.K., E.A., S.S. and F.P.; Writing—review and editing, N.K., E.A., F.P. and J.Y.

**Funding:** This research was carried out within the framework of French projects namely: (i)—PEPS-Urban mine-2014 (CNRS-INSIS); (ii)—"Investissements d'avenir"—ANR-10-LABX-21-01/LABEX RESSOURCES21; (iii)—ANR CARNOT programme.

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

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*


*2.5. Tangeretin Treatment Modestly Reduces the CD44*+/*CD24*− *Population Size*

MDA-MB-231 and MCF-7 cells were treated with tangeretin, and we analyzed the BCSC marker-expressing CD44+/CD24− subpopulation. Tangeretin modestly reduced the CD44+/CD24− cell population size from 84.1% to 56.8% in MDA-MB-231 cells and from 2.2% to 0.9% in MCF-7 cells (Figure 6). This result shows that tangeretin reduces the frequency of a BCSC trait.

**Figure 6.** The effect of tangeretin on the CD44high/CD24low cell proportion. The CD44high/CD24low subpopulation within an MDA-MB-231 and MCF-7 cell population treated with tangeretin (80 μM) or DMSO for 24 h was analyzed by flow cytometry. For flow cytometry analysis, 2 × 104 cells were acquired. The gating was based on the binding of an antibody without tangeretin (red cross). The data from triplicate experiments are represented as the mean ± SD; \*\* *p* < 0.01.

#### *2.6. Tangeretin Inhibits the Stat3 Signaling Pathway and Reduces the Sox2 Level in Mammospheres*

To investigate the cellular mechanism by which tangeretin inhibits mammosphere formation, we assessed the expression levels of Stat3 and pStat3 in mammospheres. Our results showed that tangeretin decreased the total protein levels of Stat3 and pStat3 in BCSCs (Figure 7A). The protein level of phospho-Stat3 was significantly reduced in the cytosol and nucleus of mammosphere cells. The Stat3 protein level was also decreased, as shown in Figure 7B. Additionally, we investigated Stat3 probe DNA binding to tangeretin-treated nuclear extracts by EMSA. We examined Stat3 probe DNA binding to mammosphere nuclear proteins using a Stat probe. Tangeretin reduced Stat3 DNA binding (Figure 7C, # 3). The specificity of Stat3 binding was determined using a self-competitor (100×) (Figure 7C, # 4) or a mutated Stat oligo (100×) (Figure 7C, # 5). The band indicated by arrows is Stat3 and the specific DNA complex. To examine the effect on Stat3 on mammosphere formation, we performed mammosphere formation using siRNA of Stat3. Our data showed that Stat3 reduction decreased mammosphere formation (Figure 7D). To analyze the cellular function of tangeretin, after tangeretin treatment, we checked the Sox2 level because Stat3 dimer activated Sox2 gene [18]. Our data showed that tangeretin reduced the level of transcripts and protein of Sox2 (Figure 7E). Sox2 plays a role in CSC progression [19]. Our data showed that the Stat3/Sox2 signal is important for mammosphere formation. Our data showed that tangeretin, which disregulates the Stat3/Sox2 signaling pathway, was essential for inhibiting the proliferation of BCSCs (Figure 8).

**Figure 7.** Tangeretin regulates Stat3 signaling and Sox2 regulation. (**A**) The total levels of Stat3 and pStat3 were measured in mammospheres composed of MDA-MB-231 cells and MCF-7 cells after treatment with tangeretin (0 or 60 μM) for 48 h using western blotting. Total lysates were subjected to immunoblot analysis with anti-Stat3 and anti-pStat3 antibodies. β-actin was used as an internal control. (**B**) The levels of Stat3 and pStat3 in the cytosolic and nuclear protein fractions were measured in mammospheres composed of MDA-MB-231 cells after treatment with tangeretin for 48 h using western blotting. Nuclear and cytosolic proteins were run on SDS-PAGE gels, followed by immunoblotting with anti-Stat3, anti-pStat3, anti-β-actin and anti-Lamin B antibodies. (**C**) EMSA was used to analyze mammosphere nuclear proteins after treatment with tangeretin. Nuclear extracts were reacted with a Stat3 probe and subjected to 6% native PAGE. Lane 1: Stat3 probe; lane 2: nuclear extracts with the Stat3 probe; lane 3: tangeretin-treated nuclear proteins with the Stat3 probe; lane 4: nuclear proteins incubated with a self-competitor oligo (100×); and lane 5: nuclear extracts incubated with a mutated-stat3 probe (100×). The arrow indicates the DNA/Stat3 complex in the mammosphere nuclear lysates. (**D**) Effect of Stat3 on mammosphere formation using siRNA of Stat3. Stat3-downregulated MDA-MB-231 cells were cultured for seven days using mammosphere media. Images were obtained by microscrope at 100× magnification. (**E**) The transcriptional level of the Sox2 gene in MDA-MB-231 was determined in tangeretin-treated mammospheres. A Sox2-specific primer was used for real-time RT-qPCR. Western blot analysis of mammosphere under tangeretin. β-actin served as an internal control. The data are presented as the mean ± SD of three independent experiments. \*\* *p* < 0.01 versus the DMSO-treated control group.

**Figure 8.** The proposed model for CSC death induced by tangeretin is shown.

#### **3. Discussion**

It has been postulated that high consumption of fruits can prevent more than 20% of all cancer cases [20]. This preventive effect is predominantly mediated by phytochemicals interacting with specific target proteins that play important roles in cancer [21–23]. Citrus, one of the most important food sources of phytochemicals with health benefits, has many biological properties and controls key pathways involved in pathologies such as cancer [24–26]. The combination CD44+/CD24− has emerged as the most important marker for BCSC isolation, and the CD24 population of MDA-MB-231 is low [27].

First, we purified a CSC inhibitor from citrus. Assay-based fractionation and several chromatographic methods isolated one compound from a citrus powder, tangeretin. Tangeretin is the major flavonoid of citrus. It also has antioxidant, anti-inflammatory and anticancer properties [17]. Tangeretin modestly induces apoptosis in bladder cancer cells through mitochondrial dysfunction [28]. Tangeretin and nobiletin induces G1 cell cycle arrest but not apoptosis in breast and colon cancers [29]. Nobiletin inhibits CD36-dependent tumor angiogenesis, migration, invasion and sphere formation through the CD36/Stat3/Nf-Kb signaling axis [30,31]. Quercetin suppresses breast CSCs through its inhibition of the PI3K/Akt/mTOR signaling pathway [32]. Despite numerous studies, there are no studies on tangeretin-induced antiproliferative and anti-CSC effects. Our results showed that tangeretin suppresses the proliferation of BCSCs. Tangeretin inhibited mammosphere formation in breast cancer cells (Figure 3) and modestly induced apoptosis in CSCs (Figure 4).

Our data showed the reduction of CD44+/CD24− subpopulation, mammosphere formation, colony formation and tumor formation. It is known that tangeretin did not inflict damaging effects sufficient to result in a reduced capacity to survive and proliferate. However, inhibition of the growth of breast cancers without inducing cancer cell death may be advantageous in treating breast tumors. Breast cancer cells resumed growth similar to untreated control within a day of tangeretin removal [29]. The Stat3 protein is essential for the maintenance of BCSCs [4]. The acetylated derivative of tangeretin (5-AcTMF) had anticancer effects on human glioblastoma multiforme cells through blockade of Stat3 signaling [33]. Extracellular IL-8 protein is a factor for BCSCs formation [13]. We investigated Stat3 signaling under tangeretin treatment. Tangeretin suppressed the total protein levels of Stat3 and pStat3. The nuclear protein levels of Stat3 and pStat3 were also decreased by tangeretin. We assessed Sox2 transcript levels in BCSCs under tangeretin treatment and confirmed that the Sox2 mRNA level was decreased in the treated samples. In addition, the protein level of Sox2 was decreased in treated cells compared with control cells (Figure 7).  The tangeretin-treated group had a smaller tumor size and lower tumor weight than the control group.

In our study, tangeretin inhibited BCSC formation and targeted BCSCs by inhibiting the Stat3/Sox2 signaling pathway. Tangeretin is a possible therapeutic agent for breast cancer and BCSCs.

#### **4. Materials and Methods**

#### *4.1. Reagents*

Silica gel 60 and TLC plates were purchased from MERK (Darmstadt, Germany) and Sephadex LH-20 was obtained from Pharmacia (Uppsala, Sweden). High-pressure liquid chromatography was performed with a Shimadzu application system (Shimadzu, Kyoto, Japan). Cell viability was measured using the EZ-Cytox Cell Viability Assay Kit (DoGenBio, Seoul, Korea). Tangeretin was obtained from ChemFaces Co. (Hubei, China).

#### *4.2. Plant Materials*

Citrus peel was collected from Jeju Island, South Korea. The citrus peel was freeze-dried, and the dried citrus was ground. The samples (No. 2017\_030) were deposited in the Department of Biomaterials, Jeju National University, JeJu-Si, South Korea.

#### *4.3. Extraction and Isolation of an Inhibitor*

Citrus powder was extracted with 100% methanol. The bioassay-based isolation protocol is summarized in Figure 1A. The methanol extracts were mixed with water, and the methanol was evaporated. The water-suspended samples were extracted with equal volumes of ethyl acetate. The ethyl acetate-concentrated part was loaded onto a silica gel column (3 × 35 cm) and fractionated with solvent (chloroform-methanol, 20:1) (Figure S1). The three parts were divided and assayed by evaluating mammosphere formation. The #2 part potentially inhibited mammosphere formation. The #2 part was loaded onto a Sephadex LH-20 open column (2.5 × 30 cm) and eluted in three fractions (Figure S2). The three parts were obtained and assayed by evaluating mammosphere formation. Part # 3 showed inhibition of mammosphere formation. Part #3 was isolated using preparatory TLC (glass plate; 20 × 20 cm) and developed in a TLC glass chamber. Individual bands were separated from the silica gel plate. Each fraction was assayed by evaluating mammosphere formation (Figure S3). The #1 fraction was loaded onto a Shimadzu HPLC instrument (Shimadzu, Tokyo, Japan). HPLC used an ODS 10 × 250 mm C18 column (flow rate; 3 mL/min). The mobile phase was water and acetonitrile. For elution, the acetonitrile proportion was initially set at 20%, increased to 80% at 20 min and finally increased to 100% at 40 min (Figure S4).

#### *4.4. Structural Analysis of the Purified Sample*

The chemical structures of the isolated compounds were determined by mass and nuclear magnetic resonance (NMR) measurements. The molecular weight was established as 372 by ESI-mass spectrometry, which showed a quasimolecular ion peak at *m*/*z* 373.3 [M + H]+ in the positive mode (Figure S9). The 1H NMR spectrum in CDCl3 exhibited signals due to four aromatic methine protons at δ 7.86 (2H) and 7.01 (2H), which are attributable to a 1,4-disubstituted benzene, one aromatic singlet methine at δ 6.59, and five methoxy groups at δ 4.09, 4.01, 3.94, 3.93 and 3.87. In the 13C NMR spectrum, there were 20 carbon peaks, including a carbonyl carbon at δ 177.3; nine sp2 quaternary carbons at δ 162.3, 161.2, 151.4, 148.4, 147.7, 144.0, 138.0, 123.8 and 114.8; five sp2 methine carbons at δ 127.7 (×2), 114.5 (×2) and 106.7; and five methoxy carbons at δ 62.2, 62.0, 61.8, 61.6 and 55.5 (Figure S5). All proton-bearing carbons were assigned by the HMQC spectrum, and the 1H-1H COSY spectrum established a partial structure, 1,4-disubstituted benzene (Figures S6, S7 and S9). The chemical structure was determined to be from the HMBC spectrum, which exhibited long-range correlations from the methine proton at δ 6.59 to the carbons at δ 177.3, 161.2, 123.8 and 114.8, and from the methine protons at δ 7.86 to the carbon at δ 161.2, implying that this compound was a polymethoxylated flavone (Figures S8 and S9). Finally, long-range correlations from the five methoxy proton peaks to the oxygenated sp2 quaternary carbons established the structure of this compound as that of tangeretin (Figure 2).

### *4.5. Culture of Breast Cancer Cells and Mammospheres*

Two breast cancer cell lines, MCF-7 and MDA-MB-231, were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM supplemented with 10% fetal bovine serum (FBS; HyClone Fisher Scientific, CA, USA) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, CA, USA). Cancer cells (5 × 104 or 1 × 104) were incubated in an ultralow-attachment 6-well plate with MammoCultTM culture medium (STEMCELL Technologies, Vancouver, BC, Canada). All cells were cultured in a humidified 5% CO2 incubator at 37 ◦C for 7 days. The formation of mammospheres was assessed by the NICE program [34]. Mammosphere formation was examined by examining mammosphere formation efficiency (MFE) (%) [35].


**2. CPH valorization routes and technological approaches into chemical platforms, fuels, and low value products**

To date, conventional valorization routes for transforming CPH to specialty chemicals occur either *via* biochemical, thermochemical, or physicochemical techniques. A combination of these techniques is also possible, given that lignocellulosic biomass usually requires pretreatment especially before biochemical conversion.

#### **2.1 Biochemical transformation of CPH into fuels**

Biochemical transformation of renewable raw materials involves the use microorganisms as catalyst to transform biomass into valuable products. It is often regarded as a cheaper approach for converting biomass to chemical, energy, and fuels. However, due to the recalcitrant nature of lignin component in biomass, the use of microbes to transform crude biomass into valuable products is often challenging and difficult. In this context, it is imperative to pretreat the biomass raw material in order to render cellulose and hemicellulose susceptible to microbial action. The pretreatment processes may be physical, thermochemical, biological, or physicochemical. The nature of pretreatment approach dictates the types of the intermediate chemical that would be obtained for further conversion to final product. The main biochemical routes that have been investigated using CPH as raw material are fermentation and anaerobic digestion.

#### *2.1.1 Anaerobic digestion of CPH*

Anaerobic digestion (AD) is a sequence of processes by which microorganisms break down biodegradable material in the absence of oxygen. AD basically occurs in three steps: decomposition or hydrolysis of biomass, followed by conversion of treated biomass to organic acids, and finally conversion of acids into methane gas. The main product of AD is biogas which contains methane, carbon dioxide, and some traces of hydrogen sulfide which is one of the main sources of renewable energy. The process also produces an aqueous mixture consisting of microorganisms involved in the degradation. Large volumes of CPH generated and its composition makes it a viable candidate for AD biogas production.

In 2018, Acosta and co-workers [12] investigated the production of methane and biogas yields from CPH and compared it to other agricultural residues, to evaluate the quality of the biomass raw material as a new feedstock for biogas production. The authors concluded that 50% of organic matter from CPH was transformed to biogas with 60% yield of methane. Dry AD was the preferred process choice for the authors because it gave the highest yields of methane and also, the operating conditions were stable [12].

In another interesting work, Antwi et al. [13] investigated the potential of valorizing CPH *via* anaerobic digestion and the impact of hydrothermal pretreatment on biogas yield. They compared the biogas yield and methane content of untreated anaerobically digested CPH to those obtained from the hydrothermally pretreated CPH at different severity levels. Based on their results they concluded that AD is an effective process of converting CPH to fuels. Furthermore, the impact of the pretreatment is diverse in that biogas yield increased for CPH treated at low severity levels up to 3.0. Hydrothermal pretreatment at severity levels above 3.0 lead to inhibitions in the AD process that lowered the biogas yield.

Several reports on the valorization of CPH or cocoa related residue to biogas *via* AD has proven that to be an effective approach, however, a form of pretreatment (physical, thermochemical, biological, or physicochemical) of the biomass is required to separate lignin from cellulose and hemicellulose [5, 14–17].

#### *2.1.2 Fermentation of CPH*

Fermentation is the conversion of sugars contained in biomass hydrosylate to specialty chemicals using microbes. The type of microbe used dictates the fermentation pathway as well as the end products. The conversion of CPH to bioethanol, bio-butatnol, and propanoic acid by fermentation reported in literature has been highlighted below.

Shet et al. [17] hydrolyzed CPH with HCl to release reducing sugars under optimized conditions (8.36% W/V of CPH, 3.6 N HCl, and 7.36 hours) using response surface methodology (RSM). The hydrosylate was neutralized using 5 N NaOH followed by fermentation to produce bioethanol. The inoculum was *Pichia stipites* at 2% V/V. After 72 hours of fermentation, bioethanol was distilled from the broth at a concentration of 2 g/L. They demonstrated that CPH to ethanol conversion is feasible and that CPH offers a cheaper and renewable feedstock for ethanol production. A similar work was done by Samah and co-workers [18], where CPH was hydrolyzed with HCl, H2SO4, and HNO3 at different concentrations (0.25, 0.50, 0.75, 1.00, and 1.25 M). They were further heated to 75 and 90°C for 2 and 4 hours. The highest glucose content of 30.7% W/V was obtained for CPH samples treated with 1.00 M of HCL at 75°C and 4 hours. The hydrosylate was then fermented using *Saccharomyces cerevisiae* for 48 hours at room temperature to obtain a maximum ethanol concentration of 17.3% V/V after 26 hours of fermentation.

Hernández-Mendoza et al., 2021 on the other hand performed alkaline hydrolysis on CPH and examined the effect of NaOH concentration, residence time, and temperature using a central composite design (CDD). The solid fraction was examined with X-ray diffraction (XRD) and scanning electron microscopy (SEM) in order to investigate morphological changes. It was further subjected to enzymatic hydrolysis which optimized the enzyme and solid loadings to convert cellulose to reducing sugars. The yeast *S. cerevisiae* was applied to ferment the hydrosylate. The optimum condition for the alkaline hydrolysis process occurred at 5% W/V NaOH

*Conventional and Unconventional Transformation of Cocoa Pod Husks into Value-Added… DOI: http://dx.doi.org/10.5772/intechopen.102606*

for 30 minutes at 120°C which led to an increased in the cellulose content of CPH to 57 ± 0.25% relative to that of the untreated sample of 27.68 ± 0.15%. SEM revealed changes in porosity and structure of CPH, whilst XRD showed increase in crystallinity. Enzymatic hydrolysis yielded 66.80 g/L of reducing sugars of which 80.74% were consumed during fermentation producing 18.06 g/L of ethanol in 24 hours. They concluded that CPH is a promising feedstock for bioethanol production [19].

Propionic acid production from CPH was reported for the first time by Sarmiento-Vásquez et al. [20]. In their work, alkaline and enzymatic treatment is conducted with 2.3% W/V NaOH and 2.4% V/V Cellic® CTec2, respectively to convert CPH to fermentable sugars such as glucose to a maximum yield of 275 mg glucose/g CPH. Subsequently 7.5 g/L CPH hydrosylate is fermented with *Propionibacterium jensenii* (DSM 20274) in the presence of 7.5 g/L of glycerol. A maximum propionic concentration of 10.28 ± 1.05 g/L after 75 hours of fermentation.

Sandesh et al. [21] successfully produced acetone, bio-butanol, and ethanol from inductive assisted H2SO4 hydrolyzed CPH using *Clostridium acetobutylicum*. A product distribution of 5.04 ± 0.32 g/L of acetone, 11.73 ± 0.84 g/L butanol, and 1.43 ± 0.04 ethanol is reported to have been obtained after 312 hours of fermentation.

These results are a demonstration of the potential of CPH as a cheap feedstock for the production of biochemicals *via* fermentation techniques after different pretreatment approaches have been applied to the CPH biomass to convert it to fermentable sugars.

#### **2.2 Thermochemical approaches**

Thermochemical biomass conversion approach involves all processes in which heat is used to transform biomass in the solid form to other states in the presence or absence of oxygen. Processes that fall under this category are direct combustion, gasification, pyrolysis, hydrothermal liquefaction, and torrefaction. This section examines how thermochemical conversion processes have been applied in CPH valorization.

#### *2.2.1 Direct combustion*

In direct combustion, biomass is burnt in ovens, kilns, fluidized bed combustors, furnaces with excess oxygen or air to obtain gases and ash. The combustion chambers are usually operated at temperature above 900°C. Gases and ash are the key products. The ash has been found to contain 40% potash which consists of 43% potassium carbonate and 27% potassium hydroxide. This is the process soap-makers in most West African countries harness potash from CPH to produce soft soap known locally as *alata samina* [22]. These CPH potash soap have been found to contain superior properties such as higher solubility, lathering capacity, cleansing power, and consistency compared with those produced with chemical KOH [23]. Furthermore, CPH ash has also been applied as fertilizer. Studies shows that replacing about 50% of conventional NPK fertilizer with CPH ash has had positive impact on nutrients uptake by maize plants and grain yield [24]. It has had similar effect on soil fertility, fruit growth, and yield in tomato production [25]. CPH ash obtained in a furnace at 650°C for 4 hours was evaluated by [26] as a heterogeneous catalyst for the transesterification of soya bean oil to biodiesel. Their results demonstrated that CPH ash is a superior catalyst for generating high yield of biodiesel with quality and engine performance close to that of diesel from petroleum.

#### *2.2.2 Pyrolysis*

In pyrolysis biomass is thermally decomposed in an inert atmosphere at elevated temperatures. The biomass is usually converted to volatile products with solid residue called char where the proportion of each fraction depends on the conditions of pyrolysis that the biomass was subjected to. The volatile fractions are usually condensed to obtain the liquid (bio-oil) and non-condensable gaseous fractions. Operating parameters such as reaction temperature, pressure, catalysts, hot vapor residence time, solid's residence time, etc., affect the overall process performance. The conditions of pyrolysis fall into three categories namely slow pyrolysis, fast pyrolysis, and flash pyrolysis. In slow pyrolysis, the temperature of the biomass is raised to about 500°C at low heating rates with long residence times. The solid char is the main product and it is the main route of producing charcoal which can used as fuel, activated carbon, soil conditioners, and feedstock for producing chemicals. On the other hand, in fast and flash pyrolysis, the liquid fraction or bio-oil is the preferred product. In fast pyrolysis, temperatures of about 500°C and short vapor residence time of about 2 seconds are typical to generate bio-oil from biomass. Flash pyrolysis is similar to fast pyrolysis except that the residence time is shorter in the former [27].

Pyrolysis is the most widely exploited biomass to liquid (BTL) conversion route in that the crude bio-oil can be directly used in boilers and turbines to generate electricity and heat as well as feedstock for synthesizing fuels, base, and fine chemicals [28]. By this technology, bio-oils that of high value and substitute for fuels from non-renewable sources can be produced [29]. Tsai and co-worker [30] demonstrated that slow pyrolysis of CPH produces bio-chars of more than 60% carbon content and a calorific value greater than 25 MJ/kg, dry basis at temperatures between 190 and 370°C for 30–120 minutes. They concluded that though this type of biochar exhibited lignite-like feature, it is not suitable for use as fuel in boilers due to the high potassium content. Several researchers have applied this process to CPH and have generated similar products [31–33].

CPH was pyrolyzed under fast pyrolysis conditions at temperatures 550–600°C by [29] for 2–4 minutes to yield 58 wt.% bio-oil, 30 wt.% biochar, and 12 wt.% noncondensable gases. Analysis of the bio-oil shows it contained a complex mixture of carboxylic acids and ketones with 9,12-octadecadienoic acid being the most abundant.

In another work by Mansur et al., the authors [2] reported the possibility of upgrading pyrolysis oil obtained from CPH *via* the use of heterogeneous catalysis. Firstly, they pyrolyzed CPH at 500°C for 50 minutes to yield pyrolysis oil which contained several chemical compounds including benzenediols, ketones, carboxylic acids, aldehydes, furans, heterocyclic aromatics, alkyl benzenes, and phenols. This oil was subjected to catalytic upgrade using ZrO2-FeOx where ketonization, selective oxidation, and demethoxylation reactions occurred and selectively yielded acetone, 2-butanone, phenol, cresol, xylenol, and ethyl phenol.

Prior to pyrolysis, it is imperative to pretreat the biomass by sun drying, oven drying to avoid moisture saturation, and mechanical comminution to increase the surface area for effective pyrolysis.

#### *2.2.3 Gasification*

Gasification is a thermochemical biomass conversion process which occurs at elevated temperatures above 700°C in a limited amount of oxygen. Usually 70–80% is transformed to synthesis gas (CO and H2) and the remainder is biochar. It is possible

*Conventional and Unconventional Transformation of Cocoa Pod Husks into Value-Added… DOI: http://dx.doi.org/10.5772/intechopen.102606*

to obtain some amount of bio-oil if the condition is favorable. To maximize the yields of synthesis gas and improve on the overall efficiency of the process, supercritical water, and catalyst is used [27]. The synthesis gas can be transformed to fuels and myriad of chemicals via Fischer-Tropsch synthesis [34]. CPH has been converted to gaseous products of varying composition by gasification. For instance, Gunasekaran et al. [35] investigated the numerical and experimental potential of CPH gasification in an open-core gasifier. According to their results, the composition of CO, H2, and CH4 in the producer gas was found to be 20–24%, 12.0–16.5%, and 2.0–3.2%, respectively for the conditions that were tested. The conversion efficiency and cold gas efficiency were determined to be 82 and 38%, respectively. Further, the predicted performance parameters and temperature distribution were found to be at par. Thus, CPH was found to be a promising raw material for an open-core gasifiers.

The application of recycle system on a CPH gasification in a fixed-bed downdraft reactor was carried out by Pranolo and co-workers [36]. The aim was to produce low tar fuel gas from CPH using recycle stream consisting of CO, H2, CO2, and CH4. They successfully reduced the tar content in the product gases up to 62% at temperatures ranging from 750 to 780°C. Therefore, the gas may be used as a substitute fuel for electricity generation.

#### *2.2.4 Physicochemical routes*

The valorization of CPH by physiochemical approach has mainly been by solid phase extraction or leaching in which solutes are removed from a solid by a liquid solvent [37]. Such processes have been applied in the extraction of phytochemicals and pectin from CPH. Phytochemicals are natural functional foods that possess a rich reservoir of bioactive components and nutraceuticals. Nutraceuticals was coined by Dr. Stephen De Felice and is a derivation from words "nutrition" and "pharmaceuticals." Phytochemicals are mainly foods or parts of foods that provide medical or health benefits including the prevention and treatment of diseases. There has been rapid increase in the consumption of plant-derived bioactive. Plants produce these chemicals to protect themselves but recent studies have shown that these chemicals can protect humans, animals, and other plants against diseases compound [38].

Rachmawaty et al., 2018 studied the extraction of bioactive components from CPH and the in vitro antifungal activity assay against the pathogenic fungus *Fusarium oxysporum*. The *F. oxysporum* is a deadly fungus that can cause diseases in nearly every agriculturally important plant. In the study, CPH was dry milled using a grinder into powder. Two solvents, acetone-water (7:3) and 70% ethanol was used to extract the phytochemicals. A solvent to sample ratio of 10:1 was used such that 200 ml of solvent was used for 20 g of CPH sample. The extract was found to contain alkaloids, flavonoids, tannins, and saponins, and triterpenoids which indicates the antimicrobial potential of the CPH extract. GC-MS analysis revealed four major components in the acetone solvent namely isopropyl myristate, benzenedicarboxylic acid, 9-octadecenoic acid (Z)-, methyl ester and octadecanoic acid, methyl ester. For the ethanol solvent however, three main components were found namely octadeca methyl-9,12-dienoate; 9-octadecenoic acid (Z)-, methyl ester; hexadecanoic acid, 15-methyl-methyl ester.

The acetone extract recorded the highest phenolic content and also a higher anti-fungal activity than the ethanol extract. Agar diffusion method was employed for antifungal testing and it showed that the extract was able to inhibit the fungal growth therefore leading to the conclusion that the CPH extract has great potential as a natural fungicide.

Pectin, a family of complex, acid-rich polysaccharides found in plant cell wall have been recovered from CPH by this approach. They have been extensively applied as gelling and stabilizer in cosmetics, food, and pharmaceuticals. They have the ability to reduce serum cholesterol, glucose, cancer incidence, and improved immune response in humans [39].

Pectin recovery from CPH was studied by Valladares-Diestra et al. [40] using citric acid hydrothermal treatment of CPH with concomitant production of xylooligosaccarides via enzymatic hydrolysis of the solid fraction after extraction. An optimum condition of 120°C, 10 minutes, and 2% W/V was employed for the recovering pectin. An amount of 19.3% of the biomass was recovered as pectin. They concluded that the prospects of implementing this novel method for the extraction of valuable chemicals such as pectin is very high.

Vriesmann et al. [39] optimized the variables affecting the nitic acid extraction of pectins from CPH using RSM. The optimum extraction condition was determined as pH 1.5, a temperature of 100°C, and time of 30 minutes. By these conditions a yield and uronic acid (UA) content (representing pectin content) of about 9.5 and 80%, respectively were predicted. However, experimental results gave a yield of 9.0 ± 0.4% and UA content of 66%. The predicted and experimental yield values were in close agreement, on the contrary, experimental UA content value was 17.5% lower than the predicted. This disparity was attributed to the low quality of the model for used the prediction. They further characterized the pectin a homogalacturonan highly esterified and acetylated one with some rhamnogalacturonan insertions.

Recently, Vriesmann and de Oliveira Petkowicz [41] compared the use of nitric acid and boiling water for the extraction of pectin. The pectins obtained from both extraction process was similar and identified as low methoxyl type. Rheological analysis suggests that both formed gels at low pH in spite of their high acetyl content therefore, the pectin can be used in acidic products.

### **3. Unconventional CPH valorization processes**

Recently, processes that are considered green have been utilized to extract bioactive chemical from biomass feedstocks. These processes are gaining popularity due to their inherent benefits such lower temperature, less activation time, and higher carbon yield. Microwave, ultrasound as well as super and subcritical fluid extraction have been applied to obtain valuable chemical from CPH and is discussed in this section.

#### **3.1 Microwave-assisted valorization of CPH**

Microwave has been utilized in recent times to extract biochemicals instead of conventional processes as uniform heating, time, and solvent savings [42–44] are the main advantages of this process. Additionally, it has been found to improve the accessibility and reactivity of cellulose when used to pretreat lignocellulosic biomass. Moreover, subsequent enzyme action is heightened [43]. This is the most widely applied unconventional process for CPH valorization.

In the work of Mashuni et al., 2020, microwave was used to assist the extraction of phenols from CPH using 85% V/V ethanol as solvent. The microwave heating power was varied from 100 to 300 W whilst the extraction time spanned 5–30 minutes. Using the Folin-Ciocalteu method with gallic acid as a standard, the total phenol

*Conventional and Unconventional Transformation of Cocoa Pod Husks into Value-Added… DOI: http://dx.doi.org/10.5772/intechopen.102606*

content of CPH was determined. The highest amount of phenol content was found to be 853.67 mg/L after 20 minutes of extraction at 200 W of microwave power. Upon characterizing the extract with GC-MS, it was revealed that the phenols present are butylhydroxytoluene; 6,6′-methylenebis(2-(tert-butyl)-4-methyl-phenol); 3-methoxy-2-((*2E*,*6E*)-3,7,11-trimethyl-dodeca-2,6,10-trienyl) phenol; and 7-hydroxy-3-(1,1-dimethylprop-2-enyl) coumarin. They concluded that microwave assisted extraction (MAE) is a promising technique for the extracting phenols quickly and efficiently [45].

Novel research was conducted by Nguyen and co-workers [42] where they extracted saponin from CPH *via* MAE using methanol as solvent. They used RSM (CCD) to identify the optimum parameters for the process. According to their findings, the optimum MAE conditions for obtaining the maximum saponin content and extraction efficiency from dried CPH were 85% methanol concentration, 40 minutes extraction time, 600 W microwave power, 6 seconds/minute irradiation time, and 50 ml/g solvent to sample ratio. The saponin content and extraction efficiency determined under these conditions were 69.9 mg escin equivalents/g dried sample and 71.1%, respectively. Thus, the CPH has a huge potential as a source of bioactive compounds for used in the nutraceutical and functional foods industries and to harness these compounds the optimum MAE conditions should be applied for best results. MAE was applied to isolate pectin from CPH using oxalic acid by Pangestu and colleagues [46]. In their work, they used RSM to investigate how pH, liquid to solid ratio (L/S), and irradiation time interact to affect the quantity of pectin isolated. A pH of 1.16, L/S of 25, and 15 minutes of irradiation were found to give a maximum yield of 9.64%. They emphasized that this route reduced the extraction time by 2–6 times. Further the L/S ratio can be minimized without considerable impact on the results. MAE was concluded as a powerful technique for isolating pectin using a cheaper and safer acidifying agent such as oxalic acid. Villota et al. [47] carbonized H3PO4 and KOH activated CPH via microwave assisted pyrolysis at 450°C for 5 minutes. The effect of H3PO4 and KOH on the activation of char from CPH and in all cases, H3PO4 activated carbon was observed to have higher yield and better textural properties (BET surface area = 1237.47 m2 /g, pore volume = 1.11 cm3 /g, and mesoporous) relative to that activated with KOH which exhibited severe material loss as well as low strength. Microwave assisted pretreatment of CPH has been applied by several researchers. A summary is presented in **Table 1**.

#### **3.2 Ultrasound-assisted valorization of CPH**

This process was implemented in the work of Hennessey-Ramos and colleagues, 2021 to extract pectin from CPH. RSM was used to determine the optimum operating conditions that is 6.0% feedstock concentration, 40 μL/g enzyme, and 18.54 hours on stream. Experiments involving three processes for extracting pectin namely acid, ultrasound-assisted and enzymatic extraction were conducted and compared. The results are summarized in **Table 2**.

From the results enzymatic extraction of pectin gave the best results for pectin yield followed by ultrasound-assisted citric acid extraction. The low GA content was attributed to duration (45 minutes) and temperature (60°C) of the process. They asserted that industrial operations above 60°C for ultrasonic assisted citric acid pectin extraction with the aim of increasing GA content would not be feasible owing to the inherent advantage of low temperature operation for such technologies. In the extraction of microcrystalline cellulose from CPH, it was pretreated with alkali followed

#### **Table 1.**

*Various microwave-assisted pretreatment of CPH.*

#### **Table 2.**

*Comparative analysis of chemical, ultrasound assisted, and enzymatic pectin processes.*

by ultrasonication. Ultrasound applied after alkaline pretreatment of the feedstock brought about cavitation action that helped to effectively remove fibril aggregates from the microcrystalline cellulose. A sonication time of 60 seconds and two cycles of the ultrasonication process considerably reduced the particle size of the microcrystalline cellulose to 280 nm [51].

#### **3.3 Super and subcritical fluid extraction of biochemicals from CPH**

Long extraction periods, low yield and quality of extracts, and loss of volatile compound are among many limitations of traditional extraction processes that has warranted the development of novel and green processes that overcome these limitations. Super critical and subcritical fluid extraction are among such processes that are considered efficient and time-economic [43, 52–54]. In a recent study on the extraction of phenols from CPH using supercritical CO2, Valadez-Carmona et al. [7] employed a Box-Behnken design to maximize the process variables that is temperature, pressure, and co-solvent. The optimum conditions obtained were 60°C, 299 bar, and 13.7% ethanol. By this approach, the extraction time was lowered even though the yield was low (0.56%), the quality of the extracts was improved whilst the loss of volatile compound was minimized. The highest total phenolic compounds (TPC) were found to be 12.97 mg GAE/g extract whereas the total antioxidant capacity was 0.213 mmol TE/g extract. These findings demonstrates that supercritical CO2 extraction is a promising technique that can be exploited for the isolation of natural

*Conventional and Unconventional Transformation of Cocoa Pod Husks into Value-Added… DOI: http://dx.doi.org/10.5772/intechopen.102606*

antioxidants from CPH for use in food, cosmetic, or pharmaceutical products. Another interesting work was published by Muñoz-Almagro and co-workers [55] where they compared conventional and subcritical water extraction of pectin from CPH. The latter process is a technique in which water provides H<sup>+</sup> and OH− ions at high pressure and temperature for dissolving both polar and non-polar compounds. At high temperatures the hydrogen bonding in water is weakened thereby decreasing the dielectric constant value and water polarity which consequently lowers the energy required for dissociation of water molecules in solute-matrix interactions and extraction efficiency is increased [55]. In the subcritical water extraction process, a pectin yield of 10.9% as opposed to 8% obtained using conventional extraction with citric acid as solvent. Characterization of the pectin showed that high molecular weight pectin (750 kDa) was preferentially extracted during the subcritical operation. These green techniques have been shown to possess high selectivity towards targeted compounds and potential for CPH valorization.

### **4. Future perspective**

Although several transformation techniques have been investigated for the conversion of CPH to valuable products, there is still a need to develop efficient and sustainable approaches for a holistic CPH biomass valorization process. In this context, the development of cutting-edge technologies that can efficiently transform these hitherto waste materials generated from cacao into useful chemicals that could potentially improve the global value chain of cacao production, is crucial and highly sort after. Although of interest, the uncontrolled co-production of char and gaseous products limits the overall yield of bio-chemicals so-obtained, and thus the overall efficiency of this approach. Being able to fractionate these lignocellulosic biomass waste into valuable chemicals in a selective fashion is highly desirable from economic and environmental considerations, but it remains a very important scientific challenging task due to scientific bottlenecks such as: (i) recalcitrance of lignocellulosic biomass to hydrolysis, often requiring high activation temperatures which are not compatible with the stability of sugars, the main components of lignocellulosic biomass waste, and (ii) high dilution ratio to prevent recombination reactions (for instance caramelization of monomeric sugars) leading to the unwanted formation of tar-like materials. In order to overcome such scientific hurdles, researchers should consider the coupling of mechano-catalytic technology to first release sugars contained in CPH, which can be achieved without the need of any solvent, translating into efficient and environmentally friendly synthesis approach, and a pyrolysis process to valorize lignin, the co-product of the CPH fractionation after the mechano-catalytic step.


**Abstract**

We have established and optimized standard operating procedures for growing and handling bacterial cultures in a shared core laboratory to support Integrative Structural Biology. The Integrative Structural Biology effort within the Biomolecular Research Center allows researchers to generate new knowledge about protein and RNA structure and function. We aim to understand how biomolecules assemble into stable structures and how structural dynamics impacts their function. Here we describe specific procedures for growing and handling bacterial cultures for overexpression and isolation of recombinant proteins, 15N/13C uniform labeling of recombinant proteins, protein isolation and purification, and analysis of protein solubility that are ideal for implementation in a shared research core laboratory that serves a multitude of diverse customers and research laboratories.

**Keywords:** protein expression, recombinant protein, isotype enrichment, core facility, protocols


**5. Conclusions**

In this study, based on the 14,692 literature data on global health research retrieved from the WOS core collection from 1996 to 2019, we conducted a scientometric analysis of the knowledge structure and knowledge field of global health research. At the same time, a visual analysis of the knowledge unit in the global health field was conducted, and a comprehensive knowledge map was drawn. Research in the global health field is extensive, involving multidisciplinary theories and methods, and its development requires the participation of researchers and new scholars in various fields. Scientific research cooperation between developed and developing countries in the global health field is particularly important and needs to be further strengthened. The mechanism of global health governance, the prevention and control of various infectious diseases, and the cultivation of professionals require long-term attention and discussion. This study provides researchers with an overview of global health through a systematic and comprehensive analysis of scientific output, core authors, significant institutions and countries, high impact journals, research cooperation networks, research topics, and emerging trends in the field of global health research. By presenting a new, comprehensive, and holistic knowledge map, this research contributes to the existing global health knowledge system. It also provides valuable guidance for researchers and related personnel to find the research direction and practice of global health.

**Author Contributions:** Conceptualization, M.W.; Methodology, X.L. and P.L.; Validation, M.W.; Formal analysis, X.L.; Data curation, P.L; Syntax modification, R.Z.; Perfect chart, Z.L.; Writing—original draft preparation, P.L.; Writing—review and editing, M.W.; Supervision, X.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Special Projects of Public Health Emergency Management for Chengdu University of Technology in 2020.

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


**16. Recent developments in diagnostic approaches for tuberculosis**

It is not easy to conduct a clinal diagnosis of tuberculosis very frequently as confirmed diagnosis requires culturing the bacteria *M. tuberculosis* in a sample from the patient [5] and which is very slow-growing. For lung diseases, we take morning sputum for culture purpose and microscopic studies. We also have to do Biopsies of the affected tissues, because that will provide us with a sample for culture and also for looking histologically for the characteristic presence of granulomas [53]. Mycobacterial culture confirms the presence of mycobacterium in given samples by microscopy analysis, and we may also draw the resistance profile i.e., whether the present strain belongs to the sensitive *M. tuberculosis* group or has resistance to some drugs that can be used to treat it. The major obstacles in culture MTB are that it is slow-growing bacteria and may take 3–4 weeks in liquid culture media [51]. The acid-fast bacilli of mycobacterial infection are detected by the microscopy analysis whereas latent tuberculosis disease is identified by immunological responses to tuberculosis antigens, i.e., i) Heaf test / Montoux: cofounded by BCG ii) Interferon Gamma Release Assays (IGRA) [48, 49]. There are some tools developed recently for the detection of drug-resistant MTB that facilitates early detection too, such GeneXpert, line prob. assay, LAMP assay etc.

#### **16.1 GeneXpert**

GeneXpert can detect mutations that cause resistance against Rifampicin. The test is a molecular TB test that detects the DNA of *Mycobacterium tuberculosis*. It uses a sputum sample and thus provides result in less than 2 hours. It can also detect the genetic mutations which are associated with drug Rifampicin resistance [65]. WHO recommended that this test should be used as the primitive diagnosis test in individuals suspected of having Multi-drug resistant TB, or HIV associated TB.

#### **16.2 Line probe assay (LPA)**

This technique also helps to detects mutations causing resistance against Rifampicin. Moreover, this assay can also detect mutations related to drug isoniazid [66]. The line probe assay (LPA), is typically based on strip technology and thus is used in the diagnosis of TB. It also detects RIF as well as Isoniazid (INH) resistance caused due to mutations in *rpo*β, and both *inhA* and *katG* genes.

#### **16.3 Loop-mediated isothermal amplification (LAMP) assay**

WHO has recommended the TB-LAMP (loop-mediated isothermal amplification) test that requires minimal laboratory infrastructure and has been evaluated as an alternative to sputum smear microscopy, which remains the most widespread test used in resource-limited environments. TB-LAMP is a unique temperatureindependent way to amplify the DNA of tuberculosis patients. It is a manual test that takes less than one hour and results can be visualized with the naked eye under UV light. The potent TB-LAMP instrument can be used at the level of the peripheral health center where microscopy is often performed. (https://www.who.int/tb/ features\_archive/TB\_LAMP/en/).

**23**

*Challenges in Drug Discovery against Tuberculosis DOI: http://dx.doi.org/10.5772/intechopen.97857*

At present treatment of tuberculosis requires more than one antibiotic with prolonged combination therapies to eradicate the infection and prevent resistance [58] and the standard therapy may include 4 antibiotics i.e., Isoniazid & Rifampicin (most effective drugs and these are given for six months and thus these two helps in killing the bacteria), Pyrazinamide & Ethambutol (given for first two months only) [67, 68]. During treatment antibiotics are required for a long period, the minimum treatment period is six months and if the patient is having CNS or bone disease it often goes on for at least 12 months [69]. The patient is asked to take four drugs for two months and then followed by two drugs for four months, and the actual dose given to the patient is decided by their body weight such as if a patient is lower than 50 kg, they get a lower

Corticosteroids are given to patients with CNS or pericardial disease because this reduces the further chances of having long term brain damage. All the cases need to be monitored and notified so that there can be a screening process of the patient's

*M. tuberculosis* is a difficult pathogen to combat and the frontline drugs currently in use are between 40 and 60 years of age. There is an urgent need of novel tuberculosis drugs, but the time to identify, develop and ultimately advance new drug regimens on the market has been extremely slow in the past decade. Organic biochemistry remains to be performed to know the mechanism of activity, to

empower lead advancement, and to ensure in vivo effectiveness [20]. Current efforts to develop drugs against tuberculosis are not enough to end the global tuberculosis epidemic. Due to the diversification and complexity of the infection for *M tuberculosis*, no model can completely define the in-vivo conditions in which mycobacteria are found in Tuberculosis patients and there is no sole standard detection condition for generating successful compounds for tuberculosis drug development*.* Recent efforts have focused on the development of whole-cell screening trials because objectivebased biochemical screens of inhibitors over the past two decades have not provided new tuberculosis drugs [68]. There are significant challenges in the discovery of antituberculosis drugs due to the nature of the causative bacteria. The lack of predictive models for the entry of compounds into mycobacteria is also a limiting factor. Several additional barriers in the development of tuberculosis drugs include: there are no well-established (PK)– (PD) paradigms, lack of validation and human-like pathology of animal models currently available for drug discovery, lack of clinical laboratories suitable for clinical trials, and the lack of adequate research funds. The biggest challenge in the development of anti-tuberculosis drugs is to reduce the duration of treatment for patients with drug-sensitive tuberculosis [18]. Noval drugs are needed to achieve this and overcome drug resistance. In addition, it should be possible to use new drugs for patients with HIV/AIDS co-infection. The present condition of tuberculosis drug development is far better than what was seen past 10–15 years ago. Howsoever, the development is still lacking behind because of the significant challenges in the drug discovery against drug-resistant tuberculosis and the shorter duration of the treatment required for tuberculosis prevention [12, 13]. We need to identify essential Tuberculosis targets based on better knowledge of the disease pathogen and physiology, develop sharp screening trials, and prepare compounds specifically designed to provide better clues for antibacterial activities [11].

dose while if the patient is above 50 kg, they get a higher dose [13].

**17. Available treatment**

close contacts as well [14, 17].

**18. Conclusion**


**9. 5G and the environment**

The introduction of 5G technology has a significant impact on the environment. 5G has the potential to exacerbate environmental issues like rising energy consumption and technological waste.

### **9.1 Benefits of 5G for the environment**

Given that the long-term effects of this new technology are unknown, the environmental impact of 5G is cause for concern. However, there may also be environmental advantages to 5G. The reduction of emissions and energy consumption is one of the most significant advantages. According to international standards, 5G should use much less power to operate than 4G, thereby transmitting more data while using less power. In 4G, for instance, 300 high-definition movies can only be downloaded with

#### **Figure 9.** *5G technology revolutionizing healthcare [72].*

one kWh of electricity; One kWh of 5G capacity can download 5000 HD movies. Data centers in 2020 were using 73 billion kWh of energy due to the increased efficiency of 5G technology [73].

5G is significantly more energy-efficient than previous generations of mobile networks, according to research conducted by the University of Zurich and Empa [74]. The study also concluded that applications like flexible working and smart grid technology that uses 5G have a lot of potentials to save energy and protect the environment [74]. Together with the increased use of environmentally friendly energy sources, real-time monitoring of the built environment has the potential to cut carbon emissions by 67.9 million metric tons [75]. This reduction will be made possible by energy grids, smart meters, and energy management systems that are enabled by 5G [75].

With IoT and 5G, we can fully comprehend the impact on the environment and respond accordingly [76]. Stakeholders will be able to forecast, optimize, and measure the impact of the environment using real-time data as 5G is implemented globally [76]. Once the right infrastructure is in place, 5G may also enable a new phase of the green revolution. Experts are hopeful that the increased speed of data sensors will result in a more efficient than ever real-time energy conservation system [76].

*Perspective Chapter: 5G Enabling Technologies – Revolutionizing Transport, Environment... DOI: http://dx.doi.org/10.5772/intechopen.111671*

Although the long-term environmental effects of 5G are unknown, there may be advantages to using this new technology. Real-time monitoring of the built environment, as well as the facilitation of the green revolution, are some of the potential benefits of 5G. But it is critical to keep an eye on how 5G affects the environment and take steps to minimize any negative effects.

#### **9.2 Challenges and limitations**

Notwithstanding the advantages, there are several challenges and restrictions related to 5G and its effects on the environment. The challenges include but are not limited to:

### **10. 5G and edge computing**

Edge computing aims to move decision-making operations as close as possible to data by extending cloud computing services to the network's edge [77]. Edge computing is turning out to be progressively famous because of the developing interest in constant information handling and the need to diminish dormancy [78]. The architecture of edge computing consists of three layers: a cloud layer, an edge layer, and a device layer [78] as illustrated in **Figure 10**. Sensors, actuators, and other devices that gather data make up the device layer. Edge servers, gateways, and other data-processing devices make up the edge layer. Cloud servers, which store data and provide cloud services, make up the cloud layer [78].

#### **10.1 Benefits of edge computing**

There are several benefits to edge computing, including:

• Reduced latency: Edge computing can significantly shorten the time it takes for data to travel from the source to the processing unit by processing it locally,

**Figure 10.** *Edge computing architecture.*

resulting in faster response times [79]. This is especially important for applications like telemedicine, autonomous vehicles, and smart cities that require processing in real-time or near real-time [80].

single-edge device's failure is less likely to have an impact on the entire network, this decentralized approach can also improve system resilience.

Edge computing offers advantages which include decreased latency, reduced bandwidth consumption, enhanced data privacy and security, and increased scalability. Edge computing is an appealing option for a wide range of applications, particularly those that involve large-scale data collection and real-time processing.

### **10.2 The benefits of 5G and edge computing**

The combination of 5G and edge computing offers several key benefits, including:


**4. Results**

*Appl. Sci.* **2021**, *11*, x FOR PEER REVIEW 9 of 17

to assimilate it to an environment with high degrees of mixing.

## *4.1. Hydraulic Head Fluctuations in the Quarry and Water Exchanges with the Aquifer* Figure 6 shows the simulated hydraulic head variations in the quarry and water exchanges between the quarry and the aquifer as a function of time and pumping–discharge

m, respectively, and the tortuosity was equal to 1. The longitudinal and transverse dispersivities adopted to simulate the water behavior in the quarry were equal to 100 m in order

*4.1. Hydraulic Head Fluctuations in the Quarry and Water Exchanges with the Aquifer*

Figure 6 shows the simulated hydraulic head variations in the quarry and water exchanges between the quarry and the aquifer as a function of time and pumping–discharge operations. Positive and negative values for the exchange flowrate relate to groundwater that flowed towards the quarry and water that flowed from the quarry to the chalk aquifer, respectively. These exchange flow rates were globally inversely correlated to the hydraulic head in the quarry. When the hydraulic head in the quarry increased as a result of a water discharge from the upper reservoir, the exchange rates decreased to negative values, reflecting that water flows from the quarry to the aquifer. The opposite behavior was observed during the pumping phases when the hydraulic head decreased in the quarry. operations. Positive and negative values for the exchange flowrate relate to groundwater that flowed towards the quarry and water that flowed from the quarry to the chalk aquifer, respectively. These exchange flow rates were globally inversely correlated to the hydraulic head in the quarry. When the hydraulic head in the quarry increased as a result of a water discharge from the upper reservoir, the exchange rates decreased to negative values, reflecting that water flows from the quarry to the aquifer. The opposite behavior was observed during the pumping phases when the hydraulic head decreased in the quarry.

**Figure 6.** Hydraulic head variations in the quarry (red line) and flowrate exchange between the quarry and the chalk aquifer (blue line). Positive and negative values for the **Figure 6.** Hydraulic head variations in the quarry (red line) and flowrate exchange between the quarry and the chalk aquifer (blue line). Positive and negative values for the exchange flowrate relate to groundwater that flowed towards the quarry and water that flowed from the quarry to the chalk aquifer, respectively.

exchange flowrate relate to groundwater that flowed towards the quarry and water that flowed from the quarry to the chalk aquifer, respectively. During no-activity phases, exchange rates were positive when the hydraulic head in the quarry was located at a lower elevation than the surrounding piezometric level in the aquifer, and vice-versa. During no-activity phases, the hydraulic head in the quarry continued to vary slightly since water continued to be exchanged between the quarry and the chalk aquifer until an equilibrium was reached. For example, at the end of a pumping During no-activity phases, exchange rates were positive when the hydraulic head in the quarry was located at a lower elevation than the surrounding piezometric level in the aquifer, and vice-versa. During no-activity phases, the hydraulic head in the quarry continued to vary slightly since water continued to be exchanged between the quarry and the chalk aquifer until an equilibrium was reached. For example, at the end of a pumping period, the hydraulic head in the quarry was lower than the surrounding piezometric level in the aquifer. Consequently, the chalk aquifer was drained by the quarry. The exchange rates were positive but decreased slowly, and the hydraulic head in the quarry increased slowly.

period, the hydraulic head in the quarry was lower than the surrounding piezometric level in the aquifer. Consequently, the chalk aquifer was drained by the quarry. The exchange rates were positive but decreased slowly, and the hydraulic head in the quarry In the long term, the average elevation around which the hydraulic head fluctuates tended to be equal to the elevation of the hydraulic head in natural conditions. This fact In the long term, the average elevation around which the hydraulic head fluctuates tended to be equal to the elevation of the hydraulic head in natural conditions. This fact was better observed in the regular scenarios than in the random one because of its more chaotic behavior (Figure 3). This phenomenon of gradual increase in the hydraulic head occurred because the simulated scenarios started with a pumping phase. During this initial phase, the hydraulic head in the quarry fell below the elevation of the hydraulic head in natural conditions. As a result, groundwater inflows from the chalk aquifer to the quarry

was better observed in the regular scenarios than in the random one because of its more

tial phase, the hydraulic head in the quarry fell below the elevation of the hydraulic head

contributed to increase the hydraulic head. This behavior differed to that expected in the case of an isolated lower reservoir where the hydraulic head remained constant and was not influenced by the groundwater exchanges. During discharge phases, when the volume of water previously pumped is discharged into the quarry, the hydraulic head in the quarry increased above its natural elevation, and thus, water was forced to flow from the quarry to the chalk aquifer. However, total water inflows from the aquifer to the quarry were generally greater than losses, as long as the average hydraulic head was below the natural level. Water inflows and outflows were not equilibrated until the hydraulic head oscillated around its natural elevation. This behavior must be considered, especially if there are constraints concerning the maximum hydraulic head to be reached in the quarry such as stability problems related with the elevation of the hydraulic head. Moreover, the gradual increase in the average hydraulic head was also critical because it may affect the balance between energy stored and produced by the system since the efficiency of the pumps and turbines depends on the difference in the hydraulic head between the quarry and the upper reservoir [8].

Table 3 summarizes the results from the simulations regarding the quarry–aquifer exchanges and the hydraulic head fluctuations in the quarry. The amplitude of the hydraulic head fluctuations was about 3 m, despite the large upper reservoir considered. Natural piezometric head fluctuations in the aquifer were 1 to 4 m depending on where you were in the aquifer. Concerning the maximum water exchange rates between the quarry and the chalk aquifer, they reached up to 0.53 m3/s. Slight differences in results were observed for the different pumping–discharge scenarios considered. The maximum variations in the hydraulic head in the requested quarry were less important for the random scenario than for the seasonal scenarios. The difference between them was about 20 cm. This was related with the higher frequency of pumping–discharge cycles of the random scenario since pumped and discharged volumes of water per phase are less important. As a result, fluctuations of the hydraulic head in the quarry as well as the exchange rates with the aquifer were smaller than in the seasonal scenarios. The maximum water exchange rates, which were similar for the three seasonal scenarios, were about 0.5 m3/s, whilst they were about 27% lower for the random scenario.

**Table 3.** Summary of the results concerning the quarry–groundwater interactions and hydraulic head fluctuations in the quarry during the simulations. These data concern the minimum and maximum hydraulic head in the quarry, the maximum variation in the hydraulic head in the quarry, and the maximum exchange flowrate between the quarry and the aquifer for each scenario.


**1. Introduction**

The field of applied ontology came to prominence in the 1990s [1,2], driven by knowledge engineering issues. In order to achieve coherent and shareable knowledge bases, engineers and scientists sought answers to questions in essence like "what is the meaning of a physical quantity?" [3] or "what exactly constitutes a gene?" [4]. The similarity to questions raised in philosophical ontology was readily apparent [5], opening up the possibility of joint engineering and philosophical ontology research.

Arguably, applied ontology is not merely the application of philosophy *to* other disciplines like engineering. Rather, the discipline also informs philosophy itself—at the very least by raising new questions about the existence of things, but hopefully also by novel approaches and answers to those questions. As such, applied ontology is one pathway in the journey of reconstituting a natural philosophy, in the sense of Dodig-Crnkovic and Schroeder's connected conceptual engineering [6].

The interaction with philosophical ontology is especially visible at the foundational ontology level. Foundational ontologies1 deal with fundamental aspects of our world, such as "material objects", "events", "being part of" and so forth. They underpin ontologies of particular domains such as biomedicine or information systems [2,7] (pp. 115–139).

Currently, one of the most prominent foundational ontologies is the Basic Formal Ontology (BFO) [8]. BFO is a realist ontology, based on a relatively small set of universals and relations2. That is, BFO asserts that the (real) universe can be carved up into universals such as *objects*, *processes*,

1 Also called "upper ontologies".

2 BFO includes relations (as per the specification), but these are as yet not included in the bfo.owl file. The discussion here refers to the specification.

*boundaries* and *qualities*. By "universals", BFO means " ... what all members of a natural class or natural kind such as a *cell*, or *organism*, or *lipid*, or *heart* have in common ... not only in the realm of natural objects such as enzymes and chromosomes, but also in the realm of material artifacts such as flasks and syringes, and also in the realm of information artifacts such as currency notes and scientific publications" [9] (p. 13). BFO has been successful in supporting engineering and scientific research in areas such as genetics, information systems and defence [10].

In previous work on the ontology of competitive intelligence [11], I developed a core ontology based on particularized relations, or *tropes*, which are fundamentally di fferent from the universals that BFO is based on (discussed in detail below). It is beyond the scope of this article to review the arguments for and against tropes (of which there are many flavors), su ffice it to say that tropes are prominent in ontological theory that addresses the nature and quantity of properties (like being "a chair" or "being red") [12]. At the applied level, tropes as particularized relations provide a way to connect causes to the structural relations that define entities, providing a seamless foundation for both entities and causality [11]. From either perspective, universals vs. tropes is a core ontological commitment.

The previous work compared the trope ontology to several other ontologies, but not BFO. Given the prominence of BFO, it is important for lesser known ontologies such as the trope ontology to provide at least some comparison, but preferably a "matching" to BFO. This is important because foundational ontologies often act as the "glue" to connect disparate domain ontologies and, if a matching is possible, it would link the trope ontology to the wider world of ontologies based on BFO. Moreover, given the subject matter of foundational ontologies, discussion of such matchings informs philosophical ontology—albeit with a constructive, rather than critical emphasis.

Matching is commonly defined as "the process of finding relationships or correspondences between entities of di fferent ontologies" [13] (p. 39). I include in this looking for points of similarity, where terms in one ontology can be coherently defined in terms of another. As such, "matching" reflects a perspectivist stance towards ontology. That is, it recognizes that di fferent ontologies may reflect di fferent aspects or viewpoints of reality (including perhaps some level of denial of that reality).

In summary, this article examines how the trope ontology was matched to BFO and what ontological insights we might obtain from such a matching. The next section will briefly outline the trope ontology and the remaining sections will match core terms of the trope ontology to BFO. In the conclusion, I will provide some general observations about the results of the matching and the relationship between the two ontologies.