\r\n\tIt is a relatively simple process and a standard tool in any industry. Because of the versatility of the titration techniques, nearly all aspects of society depend on various forms of titration to analyze key chemical compounds.
\r\n\tThe aims of this book is to provide the reader with an up-to-date coverage of experimental and theoretical aspects related to titration techniques used in environmental, pharmaceutical, biomedical and food sciences.
Since 1962 when the first Clark’s biosensor was introduced [1], enzymatic electrochemical devices have attracted increasing attention, recently being regarded as a powerful tool for the development of emerging wearable bioelectronics [2]. Integrating enzymes with electrochemical transduction units is one of the most popular and well-built bioelectronic systems due to outstanding selectivity and natural behaviors of enzymes [2, 3, 4]. Employing enzymes, as a catalytic system, in order to substitute nonselective metal catalysts, is interesting. Because of inherent behaviors of enzymes, enzyme-based bioelectronics offers favorable operations under mild physiological conditions of pH and temperature, unlike nonenzymatic approaches [5, 6]. In addition, enzymes will usually catalyze only one particular reaction. Therefore, such enzyme specificity enables bioelectronics to operate selectively even in complex solutions, including biofluids. Recently, there is an increasing interest in transforming traditional enzymatic bioelectronics into modern wearable platforms. Wearable enzyme electronics expands appealing spectra of a variety of applicable fields, ranging from personalized healthcare, fitness, to the environment. These applications comprise of noninvasive diagnosis of biomarkers in biofluids, such as sweat, and the monitoring of the surrounding of the wearer. Besides, electron collectors can be functionalized with enzymes to develop BFCs for energy and self-powered applications. These biodevices employ enzymes to obtain electrocatalytic oxidations of biofuels, such as glucose and lactate. This aims to achieve next-generation energy autonomy for the whole wearable system. In addition to energy-harvesting purposes, BFCs can also act as self-powered electrochemical sensors. Three main applications of enzyme-based electrodes, including biosensors, biofuel cells (BFCs), and self-powered sensors, along with their relevant aspects, will be discussed (Figure 1). An enzymatic biosensor employs an enzyme, immobilized on an electrochemical transducer, to recognize and react with the target, generating a readable electrical signal (Figure 1B). A BFC energy harvester can convert chemical energy into electricity and power wearable devices (Figure 1C) [7]. A BFC can also be designed to act as a self-powered sensor by displaying power signals proportional to the target concentration (Figure 1D) [8, 9].
\n(A) Skin-worn enzyme-based electrochemical devices. The soft electrode platform is functionalized with enzymes, allowing various applications, including (B) biosensors, (C) energy-harvesting biofuel cells, and (D) self-powered biosensors.
Skin-worn enzyme-based electrochemical devices are among the most significant wearables because the skin offers the largest organ interface and unique opportunities to be accessed noninvasively [10, 11, 12, 13]. The large epidermal area also provides sweat, which contains a variety of biomarker-rich information, such as levels of glucose, lactate, hormone, urea, pH, and electrolytes. Advantageously, skin-worn electrochemical devices can be attached directly close to the location of sweat generation, enabling the fast access for monitoring or energy harvesting before the unwanted biodegradation. In addition to physical parameters obtained from existing skin-worn biodevices (such as temperature and heartbeat), chemical data is also crucial to step further to understand comprehensive insights of individual [14]. The history of sweat content analysis began many decades ago with the development of cystic fibrosis diagnosis [15]. Establishing new “lab-on-skin” electrochemical devices enables noninvasive detection of such biometrics, essential for health monitoring and early disease diagnosis. In addition, such wearable electrochemical tools are also helpful for drug testing and chemical threat screening, such as in sports [12] and in the surrounding environment [16]. Importantly, for emerging energy technologies, sweat also contains relevant biofuels, such as glucose and lactate; this is useful to BFCs as energy-harvesting and self-powered devices, which exemplify new exciting wearable autonomous bioelectronic systems.
\nAlthough researchers are battling to create new enzymatic bioelectronics, there is a continuing need for further development. Revolutionizing traditional electrodes toward wearable bioelectronics needs careful engineering to address several key challenges associated with electrochemistry, the integration of biocatalysts, mechanical stability, environment effects (e.g., O2 fluctuations), and sweat extraction. Therefore, the bulk of this chapter will focus on examples of progress in skin-worn enzymatic electrochemical devices. Key working principles and opportunities of biosensors and BFCs will be described. In addition, perspectives emphasizing on main challenges will be discussed. The outlooks of emerging wearable electrochemical technologies will also be concluded.
\nWearable enzymatic electrochemical biosensors utilize enzymes, which are functionalized in spatial contact with electrochemical transduction units. In principle, biosensors consist of electrodes and enzyme receptors, allowing the specific binding capabilities and catalytic activity to target analytes. Interfacing enzymes with electrodes will be discussed further in Section 3.3. It should be remarked that the key consideration to fabricate a successful biosensor for nonspecialist wearers is choosing highly specific biocatalysts. Enzymatic biosensors can also function continuously because enzymes are not consumed in reactions, offering an advantage for wearable sensors.
\nEnzymatic biosensors are based on numerous mechanisms. The popular mechanism relies on the conversion of the analyte as an enzymatic substrate into a product, enabling the detection by using electrochemical transducer. Another way is to monitor the analyte (e.g., a toxic compound) that acts as an enzyme inhibitor. In addition, the enzyme can be used as a labeling transducer for bioaffinity recognition. Besides, a reverse approach can be designed to detect the enzyme level. In this case, the enzyme acts as an analyte, while the substrate is immobilized on the electrode surface. When the enzyme reaches the electrode sensor, it will generate the signal, corresponding to the concentration level of the enzyme target.
\nIn recent decades, enzymatic biosensors have been proven to be modern wearables to monitor numerous analytes, such as glucose, lactate, alcohol, and organophosphate nerve agents. Among several enzymes, oxidoreductase and hydrolase, such as glucose oxidase (GOx), lactate oxidase (LOx), alcohol oxidase (AOx), and organophosphorus hydrolase, are predominant for wearable biosensing applications. A temporary tattoo with the integration of transdermal enzymatic glucose biosensor has been introduced since glucose is a key biomarker for diabetes mellitus, which still affects hundreds of millions of patients globally (Figure 2A) [17]. The iontophoretic ISF extraction system was coupled with the amperometric detection to extract the sample containing glucose. The glucose biosensor, located near the negative iontophoretic electrode, relied on GOx immobilization on the Prussian blue (PB)-carbon electrode; this PB facilitates the electroreduction of H2O2 product, generated by the GOx reaction. The amperometric reduction of H2O2 could be detected at a potential of −0.1 V versus Ag/AgCl. The iontophoresis strategy will be discussed in Section 3.5. Additionally, the tattoo-based alcohol sensor was also invented (Figure 2B). The AOx-/PB-based sensor was designed to be close to the positive iontophoretic electrode to determine ethanol in sweat induced by transdermal delivery of the pilocarpine drug [18]. Moreover, recent efforts have been made to combine these two concepts, including glucose and alcohol sensors, on a single tattoo [19]. This holds a possibility for multianalyte sweat analysis.
\nSkin-worn enzyme-based electrochemical biosensors. (A) Transdermal tattoo-based glucose sensors, coupled with reversed iontophoresis [17]. Adapted with permission from ref [17]. Copyright 2015 American Chemical Society. (B) Tattoo-based alcohol biosensors, coupled with pilocarpine iontophoresis and wireless electronics [18]. Adapted with permission from ref [18]. Copyright 2016 American Chemical Society. (C) Biosensors integrated with a microfluidic patch for sweat collection and analysis [20]. Adapted with permission from ref [20]. Copyright 2017 American Chemical Society. (D) Microneedle-based β-lactam sensors [22]. Adapted with permission from ref [22]. Copyright 2019 American Chemical Society. (E) Integrated glucose/lactate enzymatic biosensors with electrolyte and temperature sensors. (F) Integrated sweat monitoring biosensing and transdermal drug delivery system.
Skin-worn microfluidic devices can enable the continuous flow of renewed sweat over operational periods. This addresses the challenge of mixing and carry-over between new and old sweat. Figure 2C shows an example of sweat collection microfluidic devices, coupled with glucose and lactate biosensors [20]. This offers wearable effective continuous sweat sampling and flow electroanalysis.
\nFurthermore, minimally invasive microneedles for continuous glucose monitoring have been demonstrated. For example, a GOx/tetrathiafulvalene microneedlebased amperometric sensor (~1.2 mm needle height) could be used for in vivo studies [21]. The data were also validated with the finger-prick technique, indicating a promising alternative for on-skin analysis. In addition, a minimally-invasive microneedle-based potentiometric sensor for tracking β-lactam antibiotic concentrations in vivo and real time was demonstrated Figure 2C [22]. This example represents a possibility to tailor individual therapy with the optimal efficacy.
\nMoreover, reading several parameters can complete a clear picture of individual health. A fully integrated sensor array for sweat analysis was demonstrated (Figure 2E) [23]. These integrated sensors can monitor information of glucose, lactate, electrolytes (e.g., sodium and potassium ions), and temperature. The temperature sensor is also helpful to standardize the biosensing amperometric response. Furthermore, in order to apply the biosensor glucose device for health management, a transdermal closed-loop drug delivery integrated with a sweat-based glucose electrochemical sensor was demonstrated (Figure 2F) [24]. The sense-treat concept aimed to give feedback of transdermal administration of type 2 diabetes drugs in response to the glucose level. This idea represents a possible opportunity to overcome insulin overtreatment, helping patients to maintain their homeostasis.
\nBFCs are energy-conversion devices that utilize biocatalysts to convert chemical energy into electricity. For wearable electronics, the need to anatomically power sources has attracted many research groups to develop a BFC, as a “green” energy-harvesting alternative, in order to extract energy from metabolites present in biofluids, such as perspiration. Since glucose, lactate, and oxygen are present in physiological fluids, in general, a majority of wearable enzymatic BFCs rely on (1) the generation of electrons from glucose or lactate biofuels and (2) the electron reduction by oxidants (such as oxygen). Figure 1C shows a typical example of a glucose/O2 BFC. In principle, a glucose BFC uses GOx, functionalized on the bioanode, to catalyze the glucose oxidation reaction to generate electrons. After this oxidation process, these harvested electrons are driven through an external circuit to the biocathode compartment where such electrons are accepted by oxidant molecule (commonly O2) and, eventually, generate complete electrical work. In addition to Pt-based catalysts, multicopper oxidases such as laccase, bilirubin oxidase, and polyphenol oxidase are commonly used for electrocatalyzing oxygen-reduction reaction (ORR) in the BFC cathode [25].
\nEnzymatic BFCs represent an interesting alternative due to their unique advantages, such as outstanding selectivity and behaviors of enzymes. Unlike most traditional inorganic catalyst-based fuel cells, which require harsh conditions (such as acidic conditions or high temperatures ranging from 45°C to more than 100°C), the enzyme-based BFC can operate under mild conditions (20–40°C at neutral pH). Moreover, non-specific catalyst-based fuel cells require to separate anode and cathode chambers by a thin membrane. Unfortunately, this common use of separation membrane between the anode and the cathode compartments will be unsatisfactory for skin-worn miniaturized devices. Thanks to the nature of enzymes, utilizing high specificity of enzymatic catalysis can obviate this membrane requirement, facilitating the fabrication and applications [26]. In addition, enzyme-based BFCs can operate selectively in complex biofluids.
\nInterestingly, BFCs also offer opportunities to design self-powered biosensors (Figure 1D). For example, the power is proportional to the concentration of the fuel (also acting as analyte); self-powered output itself can determine the level of the target. This offers opportunities to eliminate external energy sources for powering potentiostat and signaling systems [9].
\nAn initial concept integrating enzymatic BFCs with skin-worn technologies represented an exciting way to scavenge bioenergy available in human perspiration (Figure 3A). This demonstrated the first epidermal tattoo-based BFC that converted sweat lactate biofuel and oxygen into electricity [27]. The lactate oxidation by LOx electrocatalyzation was mediated by tetrathiafulvalene on the carbon nanotube (CNT)-based anode, while electroreduction on the oxygen-reduction cathode relies on Pt black catalyst. This system facilitates mediated oxidation of lactate at −0.1 V with a peak potential of 0.14 V (versus Ag/AgCl). This low anodic onset potential indicates the efficient electron-donor-acceptor TTF/CNT. The successful on-body test displayed a power up to 70 μW cm−2. This idea was also established on fabrics and could power a light-emitting diode with an integrated DC-DC converter [28].
\nSkin-worn BFCs and self-powered sensors. (A) Epidermal tattoo-based lactate BFCs. (B) Stretchable glucose BFCs [29]. Adapted with permission from ref [29]. Copyright 2016 American Chemical Society. (C) Stretchable textile-based BFCs acting as self-powered biosensors [30]. Adapted with permission from ref [30]. Copyright 2016 The Royal Society of Chemistry. (D) Photoelectric BFCs. (E) Textile-based BFC-supercapacitor hybrid devices [31]. Adapted with permission from ref [31]. Copyright 2018 The Royal Society of Chemistry. (F) Built-in BFCs with transdermal iontophoresis patches.
Mechanical stability has been the focus in the development of the next-generation of skin-worn BFCs due to the multiplex mechanical movements experienced in vivo. In order to minimize cracking of the device and maintain good electrochemical performance, screen-printable stretchable inks and judicious stretchable design have been engineered (Figure 3B) [29]. Combining additional degrees of stretchability with intrinsic mechanical resiliency of soft CNT/polyurethane (PU) composite offers the desirable stretchable platform. The BFC was then functionalized on the soft electrodes, allowing good mechanical stability. This holds promise applications for on-body bioenergy fields wherein resilience toward mechanical distortions is compulsory.
\nIn addition to energy-conversion applications, BFCs can be applied further as another significant tool for wearable bioelectronics. Enzymatic BFC can serve as self-sustainable biosensors (without an extra powering device). In order to expand the spectrum of BFC applications for on-skin electroanalytical chemistry, the pioneering stretchable textile-based BFCs that can act as self-powered was demonstrated (Figure 3C) [30]. These biodevices can deliver two key functions: (1) harvesting electrical power from sweat glucose and lactate and (2) displaying signals of such metabolites. Extracted bioenergy from the wearer’s sweat can directly indicate the metabolite levels. Sock-based biodevices were successfully demonstrated on human subjects, representing a promising concept for modern wearable self-powered biosensors.
\nMaximizing the loading amount of active enzyme, mediator, and conductive materials can improve the power performance of BFCs. The high amount of such active materials can be packed by a compress. However, this strategy will affect mechanical softness. Therefore, further engineering was to fabricate island-bridge assemblies merging the high enzyme loading packed islands with stretchable serpentine bridges [34]. This combination offered a soft bioelectronic skin for harvesting a good power density of 1.2 mW cm−2. This energy was sufficient to power a Bluetooth Low Energy (BLE) radio integrated with a DC-DC converter.
\nRecently, additional efforts have been made to scavenge, improve, and store energy by hybridizing textile-based energy conversion with energy storage devices (BFCs and supercapacitors, respectively) (Figure 3E) [31]. The on-body demonstration showed that after perspiring, the supercapacitor could be charged by the BFC energy and reach a stable 0.4 V output.
\nFurthermore, a photoelectric BFC was developed to convert external light andchemical energy from wearer’s perspiration into electrical energy (Figure 3D) [32]. The anode relied on a LOx/Meldola’s blue/buckypaper electrode, while the photocathode relied on an organic polyterthiophene semiconductor, which drove a reduction reaction under illumination (wavelengths of 350 nm to over 600 nm). This system presented an attractive example of on-skin autonomous power sources and sensors.
\nAdditional efforts have been made to explore new biomedical applications of BFCs. Figure 3F shows an integrated fructose/O2 BFC patch that was conjugated with transdermal iontophoresis [33]. The current generated by the BFC was used to drive an osmotic flow from the anode to the cathode, resulting in the net ionic movement of small-molecule drug into the skin. The level of transdermal current to control the drug administration could be adjusted by connecting a thin poly(3,4-ethylenedioxythiophene)/PU resistor of a programmable resistance value.
\nYoung’s modulus of the human skin is in a range of 10–500 kPa [35, 36], while the moduli of common electronic materials, such as silicon and gold, are much higher (high GPa), indicating significant mechanical mismatch when integrating with the skin. Therefore, functionalities of non-stretchable electrodes will deteriorate after multiplex deformations commonly experienced by daily life activities. Furthermore, such rigidity and bulkiness of traditional devices also restrict the wearability and comfortability [14]. Non-compliant electrochemical devices will limit continuous long-term functions due to cracking and increasing of material resistance. This increasing of resistivity, which opposes the current flow in bioelectronics, causes poor electron communication at the enzyme-electrode interface.
\nThis major challenge of skin-integrated electronics can be addressed by exploring stretchable materials which display mechanical properties in a similar range of skin’s modulus. One approach is using polymers due to their low mechanical toughness. For example, conducting materials with high moduli can be blended with soft polydimethylsiloxane or Ecoflex materials (Young’s moduli of 0.4–3.5 MPa and 125 kPa, respectively) in order to tune the mechanical properties while keeping good electrochemical functions [37]. CNT-based materials, which are powerful for electrochemical devices [38], are used to combine with soft elastomers, such as PU and styrene-butadiene-styrene (SBS) [29, 39]. PU and SBS composites have moduli of ~700–800 kPa. As shown in Figure 3C, CNT filler (with the high-aspect ratio ∼1300) was combined with PU [30], achieving stretchable conductive electrode materials. The percolation of dispersed CNTs can facilitate the electric flow in stretchable bioelectronics. Combining the intrinsic stretchability of this engineered inks with the structural stretchability of the serpentine design allows the device to tolerate strains as high as 500% with a small effect on its electrochemical performance [29]. This concept can be expanded by adding new functionalities into electrodes. For example, platinum-decorated graphite was mixed with PU to obtain stretchable electrocatalytic materials, allowing the fabrication of stretchable electrodes for glucose biosensors [40].
\nGrowing demand of wearable technologies has stimulated the need of the development of viable energy sources. The lack of anatomically power sources becomes a key bottleneck for the progress in wearable bioelectronics. Skin-worn bioelectronics mandates the compliant and efficient energy sources to supply multitasks, including sensing and data communication. In addition to developing low-power-consuming electronic microelectronics [9, 41], there is an increasing interest in advancing bioenergy-harvesting devices. Enzymatic BFCs are attractive self-sustainable energy devices to meet this growing energy demand. For example, 0.3-V complementary metal-oxide-semiconductor (CMOS) wireless glucose or lactate biosensing systems, which consumed power of ~1.2 μW, could be powered by BFCs [9]. Nevertheless, several applications of enzymatic BFCs still have some challenges, such as low-power output. The major challenge in enzymatic BFC is faced by the electrical “wiring” of enzymes with electrodes. The difficulty of electrical wiring, referring to electron transfer, and their possible solutions will be detailed in Section 3.3.
\nCompared with traditional fuel cells, enzymatic BFCs are challenging due to their multicomponent including redox potentials of enzyme, cofactor, and mediator. This results in the typical unwanted deviation of open-circuit voltages (OCV) from their theoretical maximum values, referring to “cell voltage losses.” The redox potential for electrocatalytic oxidation at the bioanode required to be higher than that of the biocathode for reduction reaction in order to deliver a sufficient electromotive force for electron transfer between enzyme active site and mediator. The voltage difference between the formal redox potentials (E°′) of redox enzyme cofactors in the active sites, in the anode and cathode, will govern the maximum cell voltage. Parameters, including redox potential of mediator and cofactor redox potential in the enzyme, can influence the resulting potential output of BFCs. Therefore, the mediator should be carefully chosen. For example, ferrocene derivatives coimmobilized with GOx at a graphite electrode can be used for glucose sensors [42]. Nevertheless, ferrocene derivatives display high redox potentials (0.1–0.4 V versus SCE); these will cause cell voltage losses in the GOx-based BFC if they are used as anode mediators. It should be noted that the difference between the redox potentials of the enzymes wired at the anode and the cathode determines the cell voltage. An example of a successful anode mediator used in skin-worn BFCs is 1,4-naphthoquinone [30]. This quinone compound is also almost insoluble in cold water, preventing leaching during on-body operations. One challenge of using GOx on the anode is the O2 competition with a mediator, decreasing the oxidation current on the bioanode. Moreover, O2 competitive reaction on the anode can produce H2O2. This by-product can inhibit GOx activity and decrease the overall BFC performance. Therefore, catalase should be cofunctionalized to the bioanode to diminish the undesirable H2O2 [43].
\nA single-enzyme BFC can usually convert only a partial portion of biochemical energy, resulting in low current output. For instance, wearable BFCs, such as for harvesting energy from lactate sweat, commonly employ a single enzyme-based bioanode, catalyzing the oxidation of lactate to pyruvate, which only harvests two electrons. In other words, they utilize only a portion of the biofuel energy and leave most of the energy in the oxidized product. Therefore, it is interesting to harvest the total of 12 electrons in order to maximize the energy-conversion efficiency. A potential solution is to design an enzyme cascade system for complete oxidation of lactate fuel. For example, the bioinspired multienzyme catalytic cascade could complete the metabolic cycle, successfully enhancing net BFC power [44].
\nFurthermore, in order to optimize the current output, diffusion and enzyme loading should be enhanced. The engineering of specific enzyme activity and three-dimensional structure of enzymatic electrodes should be explored.
\nThe selection of enzymes is a primary subject which should be discussed. Enzymes must be selected by considering their particular reactions to target analytes or biofuels for electroanalytical monitoring and energy harvesting, respectively. One of the most predominant enzymes used to develop wearable bioelectronics is GOx from Aspergillus niger. It represents an example of commercially available biocatalyst that has good stability, substrate specificity, and electron turnover rate [3, 4, 45]. It is a powerful biorecognition element for glucose biosensors, the most widely interesting devices for diabetes health management. As shown in Figure 4 (A–C), the enzyme is immobilized on the electrode, establishing a biosensor. GOx contains two 80 kDa subunits. Each holds a tightly bound flavin adenine dinucleotide (FAD) cofactor, the important redox center which has a redox potential −0.32 V (vs Ag/AgCl) at pH 7. This redox center is crucial to transfer electrons and specifically oxidize β-D-glucose to gluconolactone. However, this FAD is shielded by the protein and a glycan structure, hindering electron exchange at the enzyme-electrode interface. Inevitably, this requires research efforts to address this roadblock [46, 47]. FAD plays an important role as a common cofactor for glucose oxidation biocatalysis. The redox process for FAD/FADH2, involving two electrons, is shown in Figure 4D, where the R group represents adenosine diphosphate and ribitol connected with the flavin. However, it is O2-dependent; accordingly, O2 fluctuations can vary the performance of this type of oxidase-based bioelectronics. Although alternative O2-independent electrodes utilized NAD-dependent electrodes can be used, they require a diffusional cofactor, not simple for wearable applications. Hence, FAD-dependent dehydrogenases are becoming interesting choices since they are O2-independent and do not depend on diffusional mediators [48, 49].
\nPrinciples of interfacing the enzyme, such as glucose oxidase (GOx), with the electrode. Different generations of strategies (A–C: first, second, and third generations) are illustrated. (D) Reactions involving the glucose oxidase biocatalyst.
The first generation of biosensors relies on quantifying O2 generation or H2O2 depletion (Figure 4A). This leads to key drawbacks, such as low dynamic range, dependency to oxygen fluctuations, and interfering effects. For instance, for glucose amperometric sensors, the detection of H2O2 at common first-generation electrodes needs the high applied detection potential where interfering compounds existing in sweat, e.g., ascorbic acid, uric acid, and some drugs, are also electroactive. Lowering the applied potential for the detection is a strategy to minimize such electroactive interferences. One approach is to incorporate electrocatalysts in wearable electrodes, such as PB or Pt [17, 40]. This offers low-potential detection of H2O2 to mitigate interference effects.
\nFurthermore, researchers have developed two strategies to wire enzymes to the electrode interface (Figure 4B and C). These include (1) mediated electron transfer (MET) and (2) direct electron transfer (this may refer to mediatorless electron transfer between the enzyme and the electrode). Such new tactics are not only useful for enzymatic biosensors but also for enzymatic BFCs which also involve bioelectrocatalysis.
\nFirst, the MET strategy utilizes a redox mediator, acting as an electron-shuttle assistant between the enzymatic active center and the electrode. The substrate level, such as glucose, can then be monitored by the redox process of the mediator. This results in the independence of oxygen and mitigating the interfering signals due to the operation at low potentials. The first consideration in electrically wiring the enzyme with the electrode is the choice of the mediator that should be close to the redox potential of the active center of the enzyme to facilitate efficient electron communication between the enzyme and the conductive electrode surface. In particular, for enzymatic BFCs, the selection of mediators is crucial to positively control the cell voltage and enhance heterogeneous electron transfer to the order of a homogeneous transfer [50]. However, challenges of using mediators, particularly for BFCs, are their stability and deviated cell voltage. In addition, biocompatibility is highly vital for skin-worn applications. In spite of the assistance of electron shuttle by redox mediators, major concerns are their biocompatibility. One possible solution is employing nanomaterials or highly biocompatible catalysts. For example, mushroom/plant extracts could be used to obtain efficient “green” bioelectrocatalytic reactions for ethanol BFCs [51].
\nSecond, direct electron transfer is an ideal goal of electrical wiring. It can be achieved by employing nanomaterials which suggest the direct electron transfer between enzyme active site and electrode. This wiring strategy is based on the shortening of the electronic contact of the enzyme and electrode (a short distance of ~1.5 nm) where the redox center of the enzyme can be regenerated directly by the electrode [52]. Therefore, this strategy can maximize the performance of bioelectronics. The engineering needs to consider the position of the active site inside the protecting protein and the conformation of the protein in order to wire the conducting materials with the redox center. This still remains the most challenging topic.
\nSeveral variables also affect the response nature of enzyme bioelectronics. Consideration of the fundamental theory of their functions will help to improve their performances. A key well-known model of enzyme behaviors is Michaelis-Menten kinetics, \n
In addition, extra membranes can be a biocompatible barrier to address challenges from biofouling and interferents, especially when electrochemical operations are made in real matrices, samples, such as sweat. A perfluorinated sulfonated membrane (Nafion®) is an example membrane, which is also easy to drop-cast. This coating membrane can protect the enzymatic layer and also prevent anionic interferents, such as ascorbate [53].
\nShelf life and operational stabilities of enzymatic electrodes are among the most critical challenges. The enzyme and active materials, such as mediators, can also leach during operations. Extensive studies have been made to improve enzyme bioelectrodes, such as by crosslinking hydrogels in the presence of the enzyme [54, 55]. Such crosslinking can entrap the enzyme to be more stable; moreover, this way enhances the loading of the enzyme, while the three-dimensional structure can facilitate the transport of analytes or biofuels, improving bioelectrode functions. Nevertheless, crosslinking enzyme or covalent binding of the enzyme can change the conformation of the enzyme and thus affect the activity [56]. Furthermore, one alternative to stabilize the enzyme electrode is the addition of stabilizers, such as polyelectrolytes, dextrans, glycerol, polyethyleneimine, and hydrophobic oils [57, 58, 59]. For instance, hydrophobic mineral oil or silicone grease can be used to minimize enzyme denaturation [58, 59]. The pasting liquid helps to lower protein mobility, maintain conformational rigidity of enzymes, and barrier to hydronium ions from acid environments. This strategy can stabilize many enzymes, such as GOx, LOx, AOx, horseradish peroxidase, amino acid oxidase, and polyphenol oxidase.
\nIncreasing enzyme loading can also improve the performance of biocatalytic devices. Employing high surface nanomaterials is useful to enhance the surface loading of the target catalyst. A graphene-based electrode is a good example platform to offer a high enzyme loading (1.1 nmol cm−2); in addition, it offers a fast heterogeneous electron transfer rate (ks) of 2.8 s−1 [60]. Moreover, CNTs, which have high conductivity and specific surface, represent an outstanding candidate nanomaterial for electrochemical wiring [38, 61]. The thin nanoscale structure can intimately incorporate with the active enzyme. Adsorption of GOx on CNTs provides the apparent ks, of 1.5 s−1 [62]. The ks of GOx at the hybrid biocomposite can be as high as 11.2 s− 1 [63]. Therefore, mediatorless bioelectrodes with excellent electron transfer could be demonstrated. Their high three-dimensional architecture also offers an enhanced loading of enzyme and/or redox mediator immobilizations. As a result, this can enlarge the current output from the biosensor or BFCs. Importantly, for BFCs, the maximized OCV and current density could be observed [43]. This BFC consists of a GOx/catalase/CNT bioanode and laccase/CNT biocathode without additional mediators. The CNT/enzyme matrix was compressed together under high hydraulic pressure (10 kN). The resulting output in an air-saturated electrolyte (200 mM glucose in 0.2 M phosphate buffer solution, pH 7 at room temperature) after 3 days displayed a high maximum OCV of 1 V. Note that the GOx/catalase/CNT bioanode and the laccase/CNT biocathode showed OCV values of −0.35 and +0.6 V, respectively.
\nImportantly, biofluids from the skin (such as sweat and extracted interstitial fluids) contain a variety of chemicals that can inhibit enzyme activity, reflecting challenges in biosensing and BFC functions in real-time on-body applications. For instance, heavy metals can be found in sweat as the body expels chemicals or balances the charges. One example is Cu2+ which has been reported as an inhibitor to deactivate the enzyme. The Cu2+ in sweat can be in a range of 1.6–16 μM [11]. 0.1 μM Cu2+ could decrease the OCV value of the glucose BFC [64]. However, this enzyme-inhibitor electrochemical behavior is analytically attractive toward the development of self-powered biosensors, such as for direct heavy metal screening or indirect cysteine monitoring. For example, cysteine prefers to bind with Cu2+ via the Cu-S bond; this superior conjugation between cysteine and Cu2+ removes metal ions from the bioanode, consequently turning on the OCV.
\nSince the O2 level in biofluids may vary, first-generation biosensors, employing O2-dependent mechanism, are subject to inaccuracy. This issue can be addressed by using fluorocarbon pasting liquids to supply internal O2 [65]. Using redox mediator as a second-generation sensor is another way to eliminate this error. Furthermore, FAD-dependent glucose dehydrogenase is an option to address O2-dependent problems due to its O2-insensitive nature, compared with GOx [49]. In addition, because of the high rate of homogeneous electron transfer rate between GOx and oxygen, GOx prefers to transfer electrons to oxygen rather than to the electrode, causing undesirable O2 competition effect [66]. Moreover, for BFCs and self-powered sensors, the commonly used ORR cathode may cause the error under anaerobic conditions. The use of Ag2O/Ag redox cathode, which does not depend on ORR, can be used to operate BFCs, mitigating the possible O2 errors [30, 67]. Note that the reduction potential of Ag2O/Ag (0.342 V vs. SHE) is close to that of O2/OH− (0.401 V vs. SHE) at pH 7. Moreover, using O2-rich cathode is another possible option to mitigate O2-deficit effects [68].
\nEach person has 2.03 million sweat glands; sweat gland densities vary broadly across the skin surface and subjects, ranging from 16 to 530 glands cm−2 [11, 13, 69]. Normally, during exercise, sweat can be secreted around 20 nL gland−1 min−1 [11]. For example, the forehead or arm can generate sweat around 3 μL cm−2 or even lower. The fluctuation of sweat rate is also related to numerous factors, such as activity intensity and hydration level. Therefore, the limited volume of sweat causes a challenge in sweat analysis and operations. This leads to the development of miniaturized skin-worn electrochemical devices that can be practical in such small dead volume. For instance, the textile-based energy-harvesting BFC requires sweat volume per area of 40 μL cm−2 to deliver steady outputs [31]. Designing a capillary chamber is a possible route for low-volume electroanalytical systems [70].
\nIn addition to a passive way to collect sweat, one strategy is an active electrical-based approach, called “iontophoresis” [71, 72]. This active strategy offers on-demand sweat generation as the device can be placed to a local skin target. There are two main approaches to extract sweat: (1) iontophoresis with pilocarpine drug and (2) reversed iontophoresis without the drug. These are attractive routes for continuous sweat analysis.
\nFirst, pilocarpine iontophoresis can be used to stimulate the sweat. In principle, a small electrical current is applied to enable the pilocarpine administration across the epidermis as illustrated in Figure 5A. For example, the tattoo-based enzymatic alcohol sensor consists of a pair of electrodes located in contact with the skin surface. Small constant current (0.2 mA cm−2) was applied through the cryogel material containing pilocarpine at the anode (positive) iontophoretic side [18]. The applied electrical force will push the pilocarpine drug, which possesses a large positive charge, to eventually enter into the skin. Such transdermal drug delivery of pilocarpine can induce the local sweat, sufficient for the subsequent electrochemical detection. In addition, interstitial fluid (ISF) located under the skin can be extracted. Without this iontophoretic strategy, it is challenging to access ISF through wearable technology.
\nElectrical-based strategies using iontophoretic electrodes to extract biofluids, including (A) pilocarpine iontophoresis and (B) reversed iontophoresis.
Second, the reversed iontophoresis without pilocarpine drug can be used to extract relevant analytes, such as glucose [17]. For instance, as presented in Figure 5B, a current (0.2 mA cm−2) is applied to extract glucose in ISF. During the reverse iontophoresis process, glucose is pulled out at the negative iontophoretic compartment. Even though glucose holds no charge, the inherent permiselective characteristic of the skin prefers to transport positive species, allowing such glucose extraction. Applying electric field on mobile electric charge can cause Coulombic force, leading to a net convective flow in the skin from the anode to cathode direction. Accordingly, dissolved analytes (e.g., glucose) are also moved toward the cathode where they can be extracted and monitored. Therefore, the glucose amperometric working electrode, adjacent to the cathodic iontophoretic side, can detect the glucose level from the extracted sample.
\nThis chapter has reviewed some examples of new trends of skin-worn enzyme-based electrochemical systems, focusing on biosensors, BFC, and self-powered sensors. The existing systems provide significant advances toward the painless and point-of-care applications and personalized electrochemical biodevices, which was not possible without such new biodevices. However, researchers still face many challenges, such as electrochemistry, electrical wiring of enzymes, enzyme behaviors, the fabrication of stretchable electrodes, O2 fluctuations in biofluids, interferences, and difficulty in sweat extraction. Moreover, the workability and reliability of biodevices can be limited due to the limited fluctuating and volume of biofluids. In order to avoid frequent recalibrations, the stability of biodevices or self-calibration systems are also important. Precise electrochemical functions for on-skin applications are still very challenging. Therefore, it is required careful attention to address all challenges in order to advance such wearable technologies.
\nAlthough main skin-worn BFCs have been driven by glucose and lactate fuels, it is interesting to explore new opportunities, such as from alcohol-based BFCs, where the bioanode can be functionalized with alcohol dehydrogenases. Future efforts may be made to expand the spectrum of current concepts. New integrated devices can be achieved by designing multifunctional sensors that can provide informative series of personalized data. This will require the incorporation of big-data analysis and Internet of things (IoT) to build up integrated networks and personalized baselines of each wearer. Big data collected from networks and individuals can then warn the user whether the body is in a healthy and equilibrium state or not. It is expected that developing new electrochemical biodevices will eventually track “fingerprints” of various pathologies and disorders. This aims toward wearable systems for early disease diagnosis. Moreover, full closed-loop concepts such as biocomputing logic gate, sensing, and therapeutic systems can also be further exploited in the integration of biosensors, BFCs, and drug delivery devices, in order to obtain both diagnostic and therapeutic applications. The next success of wearable biodevices needs the hybrid of multidiscipline, including physiological medicine, electronics, electrochemistry, bio- and nanoengineering, and computer science. These continued collaborative efforts will open fantastic opportunities for addressing current challenges and step further to create novel wearable devices and acquire comprehensive big data. Ultimately, it is expected that innovative wearable electrochemical technologies and new findings will contribute to revolutionizing diverse personalized wearables and biomedical applications.
\nThe author would like to acknowledge Hassler Bueno for proof reading.
\nBioleaching is the extraction of metals from ores using the principal components water, air and microorganism [1]. It is the extraction or mobilization of valuable (target) metal from the ore, can also be defined as a process of recovering metals from low grade ore [2, 3], with regard to solubility, bioleaching can be defined as a process of recovering soluble one from insoluble impurities after dissolving sulfide metal as soluble salt in a solution [4] that results toxics and heavy metals removed. It is isolation of metals from their ores, concentrates and mineral wastes under the influence of microorganisms leading to dissolution of metal solutions of leach liquor containing metals [5], followed by solvent extraction, stripping, ion exchange, electro wining to get pure metal.
Both bioleaching and biooxidation leads to recovery target mineral; but there is technical difference between the two technologies. Bioleaching refers to the use of bacteria, the common Thiobacillus Ferrooxidans and other bacterial as a leachant to leach sulfide minerals where the target elements remains in the solution during oxidation process, after the metal recovery the solid left behind regarded as residue and in the contrary biooxodation discard the solution after having metal values in solid phase [6, 7] microorganism also engaged in removal of radionuclides and leaching of metal that are regarded as toxic in some cases and good for bioremediation of soil, the process stops radio nucleation that result the removal of stability of target elements [7].
Bioleaching has been used for a long period of time without regarded as microbial leaching process; it has been used as early as 1000 BC when a man from metal laden recovered copper from a water, passes through copper ore deposit [8]. It was in 1556 at the mine located in Spain at Rio Tinto (Rd River) mine, slurry containing very high concentration of ferric ions leached due to the action of microorganisms [4]. Copper was precipitated from the solution obtained from this river, the very first bio mineralization process was copper dissolution, then the process continued to be developed in countries like Norway, Germany and English at different era of time, in the year 1947 heap and dump leaching was practiced that leads to the development of bacterial bioleaching process [9].
The gram-negative chemolithotroph bacterial, Thiobacillus Ferooxidans was first cultured and isolated from mine water by Colmer and Hinkel [9]. Thiobacillus Ferrooxidans is rod shaped ranging in diameter from 0.3 to 0.8 micrometers (μm), in length from 0.9 to 2 μm, 0.5 μm in width in which its movement is due to a single polar flagellum [10]. Since now this bacteria is the most studied. These bacteria were able to oxidize sulfur to sulfuric acid and ferrous to ferric in acidic environment where pH value is less than 5 [7, 10, 11]. From this point onwards the technology of bioleaching has shown tremendous growth, especially industrial coppers production, which makes annualized world copper production reach up 10% from 0.2%. It was in Chile the first industrial scale copper bioleaching plant was established in 1980 using Thiobacillus bacteria [12] large-scale production begins and bioleaching taken as main manufacturing process as any convection techniques in Chile 1984 [13]. Among the many microorganism involved, bacteria (autotrophic and heterotrophic), fungi and yeasts can be mentioned. The bacterium has these calcification based on their species as Thiobacillus Ferrooxidans, Leptospirillum Ferrooxidans, Thiobacillus Thiooxidans, Sulfolobus, but there are many classifications based on different characteristics reveled by the organisms.
Acidophilic Thiobacillus species are used to leach refractory elements like gold, they generally characterized as aerobic, acidophilic, and autotrophic used to leach sulfide minerals (copper, nickel, zinc and soon). Most common bacteria involved in bioleaching are Acidithiobacillus Ferrooxidans (Thiobacillus Ferrooxidans), Acidithiobacillus Thiooxidans, Leptospirillum Ferrooxidans, Sulpholobus Spp, Sulpholobus Thermosulphidoxidans and Sulpholobus Brierleyi. Acidithiobacillus Ferrooxidans is most vital one, which was named and characterized in 1951. Most common fungi are Aspergillus Niger and Penicillium Simplicissimum. The efficiency of bioleaching depends up on physiological requirement and capability of Thiobacillus to oxidize ferrous ion (Fe2+) and sulfur (S). There are five main species of Thiobacillus, these are Thiobacillus Thioparus, Thiobacillus Dentrificans, Thiobacillus Thiooxidans, Thiobacillus Intermedius, and Thiobacillus Ferrooxidans. On the bases of pH values for growth genus Thiobacillus can be divided into two groups, those that can grow only in neutral pH values are T. Thioparus and T. Dentrificans. The second Thiobacillus species those grow at lower pH value are T. Thiooxidans, T. intermedius, and T. Ferrooxidans.
Study of different scholars at the inceptions shows the capability of bacteria (genus Thiobacillus) to oxidize sulfur compounds to sulfuric acid; it can oxidize also range of sulfur compounds (S2−, S0, S2O4, S2O42−, SO42−) [11], followed by separation process of the iron and the bacteria Acidithiobacillus Ferrooxidans (Thiobacillus Ferrooxidans) from the solution. A. Ferrooxidans is found in drainage waters and it is commonly identified as pyrite oxidizer [14]. The bacterial (acidophile) obtain energy from inorganic sources, it grows in acidic medium that fixes carbon to the bacteria itself. Most economically important metals like iron, copper, gold, and uranium can be easily extracted by using acidophilic and chemo-litho-autotrophic microorganism. Acidithiobacillus Ferrooxidans is chemoautotrophic microorganism or acidophilic. Let see the ecology, physiology, availability and genetics of microorganism involved in bioleaching. There are three basic principles for microorganism to leach and mobilize target metals from ore concentrate – redox reaction, formation of organic and inorganic acid and finally the excretion of complexing agent (Figure 1) [4].
Image of bioleaching bacterial [4].
Here is a generalized reaction used to express biological oxidation of sulfide mineral.
MS + 2O2 → MSO4, where M is bivalent metal and reaction below show a metal sulfide directly oxidize by Acidithiobacillus Ferrooxidans to soluble metal sulfate according to the reaction.
MS + 2O2 → M+2 + SO4+2 [15].
The two majorly known mechanism in bacterial leaching are direct mechanism (involves physical contact of the organism with the insoluble sulphide) or hypothesized enzymatic reaction taking place between an attached cell and the underlying mineral surface which is independent of indirect mechanisms and it is where reduced sulfur dissolution takes place [16], it is only the direct attack of the bacteria can lead to leaching. Check the following reactions.
Indirect (involves the ferric-ferrous cycle) or it is a mechanism of sulfide oxidation involves non-specific oxidation of surfaces by Fe3+ that is generated by microorganisms that oxidize iron or oxidation of mineral by ferric ions [16]. The attached cells of bacterial oxidize the surface using either of the two mechanisms [9, 11, 14]. The reaction below shows oxidation of iron.
Minerals are broken due to the attack to their constituents, that results energy production for the microorganism. This energy production or oxidation passes through intermediates reaction processes. Two mechanisms have been proposed for the oxidation, viz. thiosulphate mechanism and polysulfide mechanism. Thiosulfate mechanism includes acid-insoluble metal sulfides like pyrite (FeS2) and molybdenite (MoS2) and polysulfide mechanism includes acid-soluble metal sulfides like chalcopyrite (CuFeS2) or galena (PbS) [15]. In thiosulfate mechanism, the attack of ferric ion on acid insoluble metal sulfides brings about solubilization via thiosulfate as an intermediate and sulfates as end product. The breaking reaction shown below.
In polysulfide mechanism, a combined attack of ferric ion and protons on acid-soluble metal sulfides causes the solubilization with sulfur as an intermediate in its elemental form which can be oxidized to sulfate by sulfur-oxidizing microbes that the reaction is shown below [17].
0.125 S8 + 1.5O2 + H2O → SO42− + 2H+ the reaction show the production of sulfuric acid results hydrogen (proton) for attacking mineral.
Fe (II) is re-oxidized to Fe (III) by iron oxidizing organisms (chemotrophic bacteria), the role of microorganisms in solubilization.
2Fe2+ + 0.5O2 + 2H+ → 2Fe3+ + H2O this reaction keep iron in ferric state that oxidize mineral.
The process of chemical attack takes place on a substrate or the mineral surface where the bacteria forms a composite and attach itself as firm as possible in order to increases maturity that finally detached and dispersed into the solution.
An important reaction mediated by Acidithiobacillus Ferrooxidans is:
Strong oxidizing agent, ferric sulfate that basically used to dissolve metal sulfide minerals, and leaching brought about by ferric sulphate is termed indirect leaching due to the absence of both oxygen and viable bacterial. Check the following leaching mechanism of reaction on several minerals.
Acidithiobacillus Ferrooxidans can convert elemental sulfur generated by indirect leaching to sulfuric acid –.
This sulfuric acid maintains the pH value at levels, which is favorable to the growth of bacteria and also helps for effective leaching of oxide minerals:
Chemolithotrophic (uses carbon for the synthesis of new cell material) bacteria can be categorized based on response to temperature as mesophiles, moderate thermoacidophiles and extreme thermoacidophiles.
Mesophiles-grows at a temperature values ranges (28°C -37°C) where Thiobacillus Ferrooxidans is able use the inorganic substrate to draw energy by oxidizing Fe (II) to Fe (III) and sulfur to sulfide and sulfate. The other mesophiles is Leptospirilium Ferrooxidans that use Thiobacillus Ferrooxidans to effect the oxidization of sulfur to sulfate. Moderate thermoacidophie-temperature values ranges (40–50°C), Sulfobacillus Thermosulfidooxidans is common one, which oxidize both sulfur and iron from energy production. This category includes Archaea and Eubacteria, and most of gram-positive microorganisms are included here. Extreme thermoacidophiles-temperature ranges 60–80°C, genera Sulfolobus, Acidanus, Metallosphaera and Sulfurococcus are in this category, [11, 18, 19]. Thermal value some time extends above the limitation values, it is due to exothermal reaction which is above the maximum growth temperature of microorganism, some microorganism genus like Archaea withstand thermal values up to 90° [19, 20].
This category is formed by closely related species that can act together with a common name given Sulfolobus. Sulfolobusa Acidocaldarius, Sulfolobus Sofataricus, Sulfolobus Brierley, and Sulfolobus Ambioalous that can oxidize Fe (II) to Fe (III) and sulfur to sulfate. Aspergillus Niger and Penicillium Simplicissimum are both used to leach sulfide minerals like copper with mobilization rate of 65% and aluminum, nickel, lead and zinc by more than 95% mobilization rate. Thiobacillus and Leptospirillum are characterized by the oxidation of sulphide minerals in acidic environment and temperature values less than 35°C, with regard to area of application these two are mostly used in dump and tank leaching of metal from sulphide based mixed ores [20, 21]. The other group of genus Sulphobacillus used under the same areas of application but relatively higher temperature up to 60°C, the temperature reaches up to 90°C in case of genera Sulpholobus and Acidianus, Organotrophic microorganisms like yeast, fungi and algae which destruct sulphide mineral and aluminum silicate, facilitate bio sorption of metals that solubilize gold, these microorganism uses carbonate and silicate ore for the extraction of metals and selective gold extraction from ore floatation and metal solution.
The two bacterial leaching namely autotrophic and heterotrophic leaching has their distinct characteristics while bioleaching process takes place, in case of autotrophic bioleaching (effective on sulfide minerals) there are two proposed mechanism of Acidithiobacillus Ferrooxidans action on sulfide minerals, first the mechanism, that the bacterial oxidize ferrous ion to ferric ion in which the bulk solution where the mineral is leached counted as indirect, this mechanism which is indirect oxidation of ferrous ions to ferric ions is exopolymeric process, both takes place on the layer where the mineral is leached. The second proposed mechanism, does not require ferrous or ferric ions, the bacteria directly oxidize the minerals by biological means having direct contact mechanism of reaction. Autotrophic leaching uses both Thiobacillus Ferooxidans and Thiobacillus Thiooxidans to leach sulfide mineral and studies shows combining the two bacterial results an increase in selectivity and rate of leaching efficiency while leaching of nickel sulfide. From the heterotrophic genus of bacteria Thiobacillus and Pseudomonas are those used to leach non-sulfide minerals and from the genus of fungi Penicillium and Aspergillus (heterotrophic fungi) are those used in leaching process, a study shows 55–60% leaching rate for nickel and cobalt, some other studies indicates that 95% and 92% leaching rate achieved while using pretreated Aspergillus Niger by ultrasound for 14 and 20 days respectively which increase its stability [4, 11, 20, 22] (Table 1).
Microorganism/ both autotrophic and heterotrophic | Ore sample |
---|---|
Aspergillus Niger, Hyphomicrobium | Flourapatite (phosphorus ore) |
Pseudomonas Oryzihabitans | Magnesite, Dolomite (magnesium ore) |
Bacillus Licheniformis | Silica |
Thiobacillus Ferrooxidans, Acidianus Brierleyi, Sulfobacillus, Thermosul Fidooxidans, Sulfolobus Rivotincti | Chalcopyrite (Low and high grade), Pyrite, Covellite |
Penicillium Simplicissimum, Penicillium Verruculosum, Aspergillus Niger, Acidithiobacillus Ferrooxidans | Iron ore, Hematite, Zinc and nickel Silicates |
Thiobacillus Thiooxidans | Pyrrhotite |
Thiobacillus Caldus | Arsenopyrite |
Metallosphaera Sedula, Sulfolobus Metallicus (BC), | Pyrite |
Paenibacillus polymyxa | Bauxite (low grade) |
Heterotrophic fungi Aspergillus and Penicillium species combined to leach low-grade nickel-cobalt oxide ores, low-grade laterite ores and spudumene (aluminosilicate), these aluminosilicate (spudumene) also leached by heterotrophic yeasts (Rhodotorula rubra), Aspergillus Niger used to leach zinc and nickel silicate [11]. Bacterial leaching can be generalized in three mechanism redoxolysis, acidolysis, complexolysis, and in case fungal leaching bioaccumulation is important mechanism. To solubilize rock phosphorous, Aspergillus Niger has been used in many occasions due to the production of organic acids with low molecular weight and phosphorous is basically essential micronutrients for the growth of bio organism, these microorganism convert insoluble phosphate to soluble, the two filamentous fungi used in phosphate leaching are Aspergillus Niger and some Penicillium, the metabolic fungal reaction produces organic acid that result the formation of acidolysis, complex and chelate [22].
The second group of bacterial genus is Leptospirillum, which is categorized in moderate thermophilic bacteria that can only oxidize ferrous ions; it is dominate iron oxidizer, which is referred as Leptospirillum Ferrooxidans (L. Ferrooxidans). Oxidation process takes place under strong acidity and temperature up to 30°C, L. Ferrooxidans has high affinity to Fe2+ and low affinity to Fe3+ which results a working condition of high Fe3+/Fe2+ ratio, when redox potential is low, L. Ferrooxidans has low growth rate at the initial stage of a mixed batch culture, a native strain of Leptospirillum Ferrooxidans used to leach zinc from low grade sulfide complex from La Silvita and La Resbalosa (Patagonia Argentina) [23]. The leach liquor itself has been a place where microorganism found, higher amount of Leptospirillum Ferriphilum were in a leach liquor, in a study conducted to leach the effect of pH on the bioleaching of a low-grade, black schist ore from Finland using Acidithiobacillus Ferrooxidans and Leptospirillum Ferrooxidans as extractant [24]. The bacteria can relatively resist high concentration of uranium, molybdenum, and silver, this is due to its affinity towards to ferrous ions or resistivity to refractory elements, but it cannot oxidize sulfur or any sulfur related compounds. By combing it with other sulfur- oxidizing acidophiles, sulfur-oxidizing process can be achieved; these are T. Caldus, T. Ferrooxidans, or T. Thiooxidans, to oxidize sulphidic gold concentrate a mixed culture of Thiobacillus and Leptospirillum has been used [11].
The third group thermophic bacteria mainly characterized by oxidation of iron to assure growth chemolithorophically, some are facultative autotrophs that require synergetic effect of other microorganism to like yeasts extract, cysteine, or glutathione. Among the microorganism in this group Sulfolobus species is the major one, these organism categorized as moderate thermophilic at an average values of temperature 40°C -60°C and the second group is extreme thermophilc at an average values of temperature 65–85°C. One of the moderate thermophilic gram positive bacteria, Sulfobacillus Thermosulfidooxidans is facultative autotrophs in which its growth stimulated by yeasts extract, where the presence of CO2, weight and volume ratio (w/v) are factor to facilitate and inhibit growth. From of extreme theremophilic Sulfolobus Acidocaldarius and Acidianus Brierleyi are those in genera Archaebacteria, among the other four genera Sulfolobus, Acidanus, Metallosphaera, and Sulfurococcus act aerobically and categorized in extreme thermophilic acidophilic bacterial which oxidizes ferrous and elemental sulfur and sulphide based minerals. These bacteria grows under all conditions (auto, mixo, heterotrphic) depending on the yeast extract ratio (w/v), found in facultative chemolithotrophic species act in acidic medium and temperature value can be up to 90°C [11].
All the major concepts of bioleaching have been discussed, so what are factors affecting rate of bioleaching and leaching efficiency, the major factors can be summarized as microbiological, mineralogical and physiochemistry factors. A physiochemistry factor includes temperature, pH, redox potential, oxygen content, carbon dioxide content which facilitate mineral oxidation required for cell growth, mass transfer, light, surface tension which mean that the topography of mineral surface that indicate the rate adsorption and crystal structure which has direct relation on the rate of reaction. Microbiological factors includes microbial diversity that is the distinct nature of micro organisms with regard to range of unicellular organisms, variety of microorganism found in an environment suitable for bioleaching, these includes bacteria, fungi, algae, flagellates, and those found in microbial biocenosis, the other microbiological factors are population diversity, metal tolerance, spatial distribution microorganism and adaptation ability of microorganism. The third major factor is the nature of mineral processed, characteristics like grain size which affect rate of dissolution, porosity related to rate of chemical attack and digestion rate, hydrophobicity is another physiochemistry factor to determine the rate leaching, hydrophobicity is differentiating whether the elements are water hating or loving while floatation takes place. Process is the other major factor affecting leaching efficiency, techniques where bioleaching process takes place (heap, dump, in situ) which we will be discussed below, pulp density is the variable which results variation on dissolution rate, a study shows that dissolution metal increases while pulp density increases but it is based on (w/v) ratio that is between 5 and 20%, the other factor is concentration of target mineral, this can inhibit the growth of microorganism, that cause a limitation of pulp density usage [25]. Stirring speed is also another factor affecting rate of dissolution and geometry of the heap during heap leaching process, the other major factor is the presence of fluoride released from the ore sample, which inhibits the process of bioleaching, and when the release decreases the rate of inhibition eventually reduced.
Besides leaching process microorganisms are used for bioremediation of mining sites, treatment of gangue, tailing, and mineral wastes from the industry, contamination of sediments due heavy metals and soil from toxicities, sewage sludge can cured by microorganism in which the process is called bioremediation [26].
The successful bioleaching process is characterize by the intimacy of microorganisms to a mineral surface, strong attachment result high rate of oxidation and dissolution on a substrate (mineral surface), this is achieved by the rate success of bio film formation. In general leaching techniques are two – Percolation leaching – a solution infiltrate through a fixed mineral location, and agitation leaching - mineral bearing ore stirred by a solution but while working in large scale, percolation leaching is usually chosen [7]. The principal commercial methods are aerated stirred-tanks, in situ, dump, heap, vat, bench scale, tank, column, reactor leaching are among the many. It was dump bioleaching process taken as the first commercial bioleaching in 1950 used to leach copper from sulfide minerals, since then bioleaching bloomed by copper oxide heap leaching, industrial microbial leaching process applied for sulfidic gold and bioheap commercial leaching of copper ore (chalcocite and covellite). The high production of bio heap leaching of copper in 1980 established at Lo Aguirre mine in Chile processing 16,000 tones ore/per year at the inception [27], these wipe the way that led to Chile’s industrial bio copper production in large scale especially from the year 1984 [28].
Aerated, stirred-tank bioreactors, used in mineral concentrate feeds, involve a series of stages that can have lots of tanks connected in parallel depending on the retention of the concentrate [7] a study conducted to check Na-chloride can possibly enhances the chemical and bacterial leaching of chalcopyrite uses three bioreactors engaged with inoculum of the bacteria [29]. Other tanks needed for value adding purposes which are usually single tanks might be connected in series, since these tanks subject to chemical attack, air, heat and sulfide mineral, they should be relatively resistant to corrosion, chemical attack, and soon, in order to have these character tanks can be lined with rubber, galvanized, or other corrosion protection method like using sacrificial anode or using high grade material like stainless steel, aluminum or copper.
Temperature maintained at optimum level by cooling coil or some time tanks are equipped with water jackets depending on the required temperature by the bacteria, these values can be conditioned based of the mineral to be leached, and sometimes the chemical used to enhance the leaching process [29]. Several tanks can be continuously arranged, named as continuous stirred tank reactor (CSTR), as per the above it can be followed by a series of small equal sized reactors [16]. Example of bioleaching of sulfidic gold concentrates, that the discharge from the final stage is subjected to water washing and solid/liquid separation in thickeners. Even though there is less power consumption basically used for agitator and blower, it has linear relationship with the amount of sulfide -sulfure which is required to oxidize and recover the target metal from the parent ore, rate of recovery depends up on the metal grade also.
The main advantages of these tank over other conventional methods like pressure autoclave, roasting, smelting, calcination and soon are; it has low capital and operational cost, relatively less construction period, less complicated requiring less skilled man power and most importantly it is environmental friendly. In general Australia, Chile, USA, Brazil and South Africa are among those countries involved in bio oxidation by stirred tank [7, 16].
Dump (run of mine) [18] leaching involves uncrushed waste rock and low grade ore is piled up or changing a pit to dump by blasting it. Conventional methods would be very expensive to process these type of ores samples, except dump leaching, dump can be very huge, containing in excess of 10 million tons of waste rock, up to 60 m deep [7, 30].
In order to digest some of unwanted minerals like silicate and to promote the growth of acidophilic microorganism, acid water solution is spread on the top surface, the acid water solution percolate through the dump, the more acidic the environment the more growth of microorganisms that oxidize minerals to be recovered. The pregnant leach liquor or acid run-off is collected at the bottom of the dump, from where it is pumped to a recovery station. After collection the process followed by solvent extraction, electro wining for the metal production but dump aeration is vital for the microorganism to growth, tailing from solvent extraction recycled on the top of dump. Escondida mine found Chile is the biggest bio dump leach in world [26, 30].
Heap leaching (crushed and agglomerated) [18] is composed of air, acid and microorganism where commutation takes place on rock samples to turn it to smaller particles which increases the surface area for acid digestions and conditioning it to microorganisms, particle should not be very fine and should be piled allowing a simplifies aeration pipe placed to facilitate air flow. To improve drainage of the mineral containing solution from the bottom of the ore, conditioned ore is spread on specially engineered pads (lined with high-density polyethylene (HDPE)), which consist of perforated plastic drain lines and air also supplied to optimize the growth of microorganism [7]. Heap can be large up to kilometer long, but commonly less than 500 m wide and 10 m long, the size and height of a heap depends up on air (for bacterial to grow) water, acid, heat generated due to the process and its dissipation [31]. Heap surface should be permeable enough for the sulfuric acid to infiltrate and dissolves iron to ferric solution producing ferric ion that react with copper sulfide results ferrous ion and copper solutions. Acidithiobacillus Ferrooxidans oxidize iron where the bacterial can be inoculated and works by attaching itself to ore, with having free movement. After collecting PLS (pregnant leach solution), then solvent extraction is followed where the target mineral recovered and formed into cathodes.
This aerobic bacteria works only in the presence of oxygen in the heap, those bacteria consume it from the solution where oxygen is in liquid phase. This process enhances the conversion of ferrous to ferric ions as per the reaction below.
Heap some time can be crushed 19 mm with rotating drum with acidified water [29] aeration can be conducted using low pressure fans those directing air through piping on the pad [26]. It is clear that heap leaching requires the preparation of the ore, primarily size reduction, so as to maximize mineral-lixiviant interaction and lay of an impermeable base to prevent lixiviant loss and pollution of water bodies. Heap leaching basically used to leach low-grade ore of copper and zinc, even in the case of copper grade level can be (0.2–2%). To have an effective heap leaching process a mathematical model has been developed by taking heat, mass transfer, liquid, gas flow and chemical process in to consideration [31]. Heap also employed to bioleach silicate mineral, in a study where two microorganism were tested ‘Ferroplasma acidarmanus or the common Acidithiobacillus ferrooxidans against the amenity of silicate minerals. Beside oxidation process energy was generated from flat plate solar energy collectors where heap is designed by HeapSim, heap bioleach simulation tool was used to simulate the heap and process occurring in the heap, even calculating the copper output [32].
In situ leaching requires making the ore permeable for a solution and air to be circulated through the ore body. It does not require metal containing material to be removed from the ground [18]. It employs a method of recovering target minerals from the leach solution. The acid solution percolates until it reaches to impermeable layer. In situ includes recovery of minerals from the intact ore. The resulting metal-enriched solutions are recovered through wells drilled below the ore body. In case of in situ leaching the main concern is pollution of ground water, with this regard there are three types of ore bodies generally considered for in situ leaching: surface deposits above the water table, surface deposits below the water table and deep deposits below the water table. It is burden materials, establish permeability allowing air to pass in which metal bearing solution collected in the sumps [7]. It is combined with mineral recovery operation time and again to pull out the minerals from recovered fluid or pregnant solution or leachant. Acidified leach solutions, applied to the top surface of the entire ore zone, infiltrate through the fragmented ore due to the blast. The leaching bacteria become established and facilitate metal extraction. Metal-rich solutions or large volume of solution is circulated with the aid of gravity flow and pumped and recovered in sumps then again pumped to the surface for metal recovery, the returning fluids to the extraction operation are known as “barren solution”. Metal recovery depends on two major things first the bacteria used (Acidithiobacillus Ferrooxidans) and permeability of the ore-body, which can be increased by fragmenting of ores in place, called “rubblizing”. Due to the ground water pollution this leaching process becomes less used and less popular [18] on the contrary it has been said that it is a best substitute for open pit and shaft mining operation, basically when in situ leaching is applied, no gangue or tailing is byproduct, it also called green mining or mine of the future [33].
Recent study shows that elements like uranium, copper, gold, zinc and other elements are commercial focus of bioleaching and biooxidation [34]. Many studies indicate microbial leaching is more important in low-grade ore, ore sample collected from Mianhuakeng uranium mine located in northern Guangdong province in China, leached by heap, by mixed microorganism of Acidithiobacillus Ferrooxidans and Leptospirillum Ferriphilum with 88.3% leaching efficiency [35]. Uranium leaching takes places by indirect mechanism, as Acidithiobacillus Ferrooxidans does not directly interact with uranium minerals. The role of Acidithiobacillus Ferrooxidans in uranium leaching is the best example of the indirect mechanism. Bacterial activity is limited to oxidation of pyrite and ferrous iron. The process involves periodic spraying or flooding of worked-out stops and tunnels of underground mines with lixiviant [4]. The pH of lixiviant was optimized during the bioleaching of uranium from low grade Indian silicate-apatite ore with 0.024% of U3O8. This study uses Acidithiobacillus Ferrooxidans for leaching and biochemically generated ferric ions as an oxidant, optimizing particle, pulp density and redox potential results 98% uranium bioleaching. In this indirect bioleaching of uranium, the bacteria generate ferric sulfate and pyrite is oxidized by a lixiviant, within acidic environment the oxidations of ferrous ion to ferric ions process executed by the bacteria is fasters than chemical oxidation [36]. In case of uranium bioleaching the main drawback is to oxidize uranium (IV) since it insoluble but on this bioleaching process when ferrous sulfate produced in the process, then re-oxidized to ferric sulfate which enzymatically oxidize uranium (IV) to uranium (VI) by the energy produced by this reaction. A case study in India at Jaduguda mines proofs that use of biogenic ferric sulfate produced by the strain which was then used for efficient uranium extraction and cause no harm to the environment, while extracting uranium, use of reduced MnO2 in Bacfox process to generate biogenic ferric sulfate, results passed air saturated ferrous sulfate solution over Acidithiobacillus Ferrooxidans which is absorbed on solid surface [36]. Since the permeability of the ore surface is a factor, the above study uses a process called “rubblizing” that increase fragmenting of ore in place which can be applied in the extraction of sulfide mineral, gold and uranium. While isolating the bacteria from mine water, the isolation media and H2SO4 consumption during isolation, pH variation and temperature were determinate factors, the microbial cell count and the growth of (A. Ferrooxidans) determines by rate of oxidation of iron from Fe2+ to Fe3+, so while leaching if the amount of Fe2+ decrease means the bacteria is using it as energy source to convert it to Fe3+, uranium bioleaching depends on the synergic effects Fe3+ and proton produced by the bacterial [37] that process uses either of the two energy sources to growth iron or sulfur. The reaction of making insoluble uranium to soluble form is as follows [38].
Studies indicate that microbial cell count and pulp density ranges 5–30% (w/v), particle size <75 μm has brought an optimum ore leaching but it should be clear that each ore has its own distinct behavior and no size fits all, meaning results indicated here might be different for another ore sample due to ore elemental composition, crystal structure, grade, topography and surface tension.
The ore is loaded on a water-resistant surface or ore is piled on an impermeable surface until a dump of suitable dimension forms. After leveling the top, then spraying a leach solution onto the dump is followed [4]. These dump is a habitat of heterogeneous microorganism. Dump can have variety particles sizes, where the bacterial annexation, which is anaerobic (microaerophilic), thermophilic begins from the top.
Dump leaching used to pretreat low-grade, refractory- sulfidic gold ores and to leach copper from chalcocite ores while ore grade is low with values ranges between 0.1–0.5%. Copper can be obtained from ore rocks from the mound then washed with dilute H2SO4 to facilitate the oxidation process of mineral by acidophiles, which is followed by cementation process where copper is precipitated from the drainage with scrap iron since it primary iron oxidizing process [39]. Check the leaching process of copper sulfide chalcocite (Cu2S), which occurs with pyrite (FeS2), leaching is due to ferric ion reacts with copper sulfide mineral processes ferrous and copper ions in solution.
In these regions indirect leaching by ferric sulphate also prevails. The exterior of the dump is at ambient temperature and undergoes changes in temperature reflecting seasonal and diurnal fluctuations. Many different microorganisms have been isolated from copper dumps, some of which have been studied in the laboratory. These include a variety of mesophilic, aerobic iron and sulfur oxidizing microorganisms; thermophilic iron and sulfur oxidizing microorganisms; and anaerobic sulphate reducing bacteria. In copper leaching the concentration of target metal by itself is an important variable, copper concentration (100–300 mM range) is values cause difficulty for the microorganism to operate, selecting the microorganism is one of the mechanisms of copper resistant, Acidithiobacillus Ferrooxidans can resist copper concentration and strong acidic environment [40]. Thiobacillus Ferrooxidans was the main product observed after a culture study, from an ore or leach solution for the identification of composition of bacterial population and incase of low ferrous ions, it was Leptospirillum Ferrooxidan was observed, the study shows that utilization of ferrous iron as energy source is dominated by the previous bacteria as the culture shows. Pseudomonas aeruginosa, where heterotrophic bacteria produce various organic acids in an appropriate culture medium is used in copper leaching [41]. The addition of salt in bioleaching of copper resulted process enhancement, after designing the bioreactor the bioleaching of copper was enhanced in both stirred tank or shack flask by adding sodium chloride in leach solution, increasing the dissolution of Fe3+ that eventually reduces precipitation [29] addition of some elements might result inhibition of bioleaching process, fluorine in solution increase the viscosity of leach liquor that result inhibition of bioleaching [42]. It is important to understand the microbiology, which is responsible or identify a means to study bulk activity of microorganism, these features are oxygen uptake in solid and liquid samples, redox potential, pH, ferrous iron concentration and temperature. Microbial leaching has also direct relation with enrichment and culture from solution of ores. Acidithiobacillus Thiooxidans, Acidithiobacillus Ferrooxidans, and Leptospirillum Ferrooxidans have been cultured where the process run at an ambient temperature and the strain of bacterial related to the microorganism mentioned here [27, 43]. Leach solutions enriched with copper exit at the base of the dump and are conveyed to a central recovery facility. In most large-scale operations the leach solution, copper-bearing solution pumped into large cementation units containing iron scrapings for cementation and then electrolysis followed [4]. It was in Chile and Australia the commercial bio heap leaching of copper started mass production. And the first bioleach heap copper extraction plant is in China [44]. The copper extracted percentage can be calculated as,
E = Copper content in the solution/copper content in the sample X 100% [41].
Acidophilic bacteria are able to oxidize gold containing sulphidic ore, such a process can be ameliorated by conventional process of cyanidation, these basically reduces the complexation by increasing the capability of microorganisms to reach to the target metal. Certain sulphidic ores containing encapsulated particles of elemental gold, resulting in improved accessibility of gold to complexation by leaching agents such as cyanide. Relative to other conventional process and pretreatments like roasting, smelting and pressure oxidation, bio-oxidation demands less cost and no harm to nature [7]. Though it is under study a commercial bio-oxidation and bio heap leaching of gold prior cyanide extraction. It is the bacteria, Acidithiobacillus Ferrooxidans used to oxidize the sulphide matrix for gold recovery. Prior to extraction, gold ore must be bio-oxidize by the bacteria. In this process refractory sulphidic gold ores contain mainly two types of sulphides: pyrite and arsenopyrite where silver ion was used as a catalyst in acidic environment. Since gold is usually finely disseminated in the sulphide matrix, the objective of biooxidation of refractory gold ores is to break the sulphide matrix by dissolution of pyrite and arsenopyrite and extract 95% of iron and arsenic, the residue of both filtered through a vacuum pump. The consumption of cynide is much higher while biooxidation, the study suggested that using thiourea instead of cyanide is much less toxic but since the process require high consumption of thiourea cost increase steadily, consumption of thiourea reduced by using different agents like SO2, bisulfite, cystine, cystine with oxygen during extraction process [45].
The mesophilic tank leaching is the most common bioleaching process in the world; thermophilc tank is favored while the temperature is high, among such tanks BioCop™ well known, In order to have effective thermophilc tank the following are basic requirements, microbial catalyzed reaction which is needed to facilitate metal dissolution by microbial oxidizing of ferrous iron to ferric iron, initial solublization of ferrous ion takes place using acid solution, oxidation of mineral sulfide takes place by the combination effects of ferric iron and acid solution followed by oxidization of reduced sulfur to sulfate by microorganisms. Reactor configuration is the other factor where the six equal size continuous reactor, three arranged in parallel considered as primary reactors, and the other three arranged in series considered as secondary reactors, in this case reactors are considers as a large continues stirred tank supplied with aeration and agitation. The other factors are oxygen, carbon dioxide, pulp density and finally even though the operational cost is much less plant location, construction material, blower or compressor to supply oxygen to the microbes, high power agitator in case of oxygen plant for oxygen dispersal in the reactor. Growth of industries results the demand of metals in very high quantity and likely go further in the years to come. This brings diminution of high grade ore with effluents and solid wastes that needs to be treated to recover the important elements and protect the environment.
Regarding to environment biohydrometallurgy is vital process, the fact that bioprocess is conducted without the presence of toxic chemical and relatively required low cost makes it most needed. The direct implication of microorganisms in the reduction of uranium is of considerable interest because of its potential application in bio remediating of contaminated sites, in pretreating radioactive wastes, bioleaching is becoming a promising technology.
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',metaTitle:"Publication Agreement - Chapters",metaDescription:"IN TECH aims to guarantee that original material is published while at the same time giving significant freedom to our authors. For that matter, we uphold a flexible copyright policy meaning that there is no transfer of copyright to the publisher and authors retain exclusive copyright to their work.\n\nWhen submitting a manuscript the Corresponding Author is required to accept the terms and conditions set forth in our Publication Agreement as follows:",metaKeywords:null,canonicalURL:"/page/publication-agreement-chapters",contentRaw:'[{"type":"htmlEditorComponent","content":"The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
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\\n\\n3. CORRESPONDING AUTHOR'S DUTIES
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\\n\\nLast updated: 2020-11-27
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The Corresponding Author (acting on behalf of all Authors) and INTECHOPEN LIMITED, incorporated and registered in England and Wales with company number 11086078 and a registered office at 5 Princes Gate Court, London, United Kingdom, SW7 2QJ conclude the following Agreement regarding the publication of a Book Chapter:
\n\n1. DEFINITIONS
\n\nCorresponding Author: The Author of the Chapter who serves as a Signatory to this Agreement. The Corresponding Author acts on behalf of any other Co-Author.
\n\nCo-Author: All other Authors of the Chapter besides the Corresponding Author.
\n\nIntechOpen: IntechOpen Ltd., the Publisher of the Book.
\n\nBook: The publication as a collection of chapters compiled by IntechOpen including the Chapter. Chapter: The original literary work created by Corresponding Author and any Co-Author that is the subject of this Agreement.
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\n\n2.1 Subject to the following Article, the Corresponding Author grants and shall ensure that each Co-Author grants, to IntechOpen, during the full term of copyright and any extensions or renewals of that term the following:
\n\nThe aforementioned licenses shall survive the expiry or termination of this Agreement for any reason.
\n\n2.2 The Corresponding Author (on their own behalf and on behalf of any Co-Author) reserves the following rights to the Chapter but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Chapter as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\n\nThe Corresponding Author confirms that they (and any Co-Author) are and will remain a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\n\nSubject to the license granted above, copyright in the Chapter and all versions of it created during IntechOpen's editing process (including the published version) is retained by the Corresponding Author and any Co-Author.
\n\nSubject to the license granted above, the Corresponding Author and any Co-Author retains patent, trademark and other intellectual property rights to the Chapter.
\n\n2.3 All rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the Corresponding Author's or any Co-Author’s specific approval.
\n\n2.4 The Corresponding Author (on their own behalf and on behalf of each Co-Author) will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Chapter as a consequence of IntechOpen's changes to the Chapter arising from translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits.
\n\n3. CORRESPONDING AUTHOR'S DUTIES
\n\n3.1 When distributing or re-publishing the Chapter, the Corresponding Author agrees to credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen. The Corresponding Author warrants that each Co-Author will also credit the Book in which the Chapter has been published as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Chapter.
\n\n3.2 When submitting the Chapter, the Corresponding Author agrees to:
\n\nThe Corresponding Author will be held responsible for the payment of the Open Access Publishing Fees.
\n\nAll payments shall be due 30 days from the date of the issued invoice. The Corresponding Author or the payer on the Corresponding Author's and Co-Authors' behalf will bear all banking and similar charges incurred.
\n\n3.3 The Corresponding Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Chapter worldwide for the full term of the above licenses, and shall provide to IntechOpen upon request the original copies of such consents for inspection (at IntechOpen's option) or photocopies of such consents.
\n\nThe Corresponding Author shall obtain written informed consent for publication from people who might recognize themselves or be identified by others (e.g. from case reports or photographs).
\n\n3.4 The Corresponding Author and any Co-Author shall respect confidentiality rights during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Corresponding Author and any Co-Author are confidential and are intended only for the recipient. The contents may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\n\n4. CORRESPONDING AUTHOR'S WARRANTY
\n\n4.1 The Corresponding Author represents and warrants that the Chapter does not and will not breach any applicable law or the rights of any third party and, specifically, that the Chapter contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy. The Corresponding Author warrants and represents that: (i) the Chapter is the original work of themselves and any Co-Author and is not copied wholly or substantially from any other work or material or any other source; (ii) the Chapter has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) they themselves and any Co-Author are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) they themselves and any Co-Author have not assigned and will not during the term of this Publication Agreement purport to assign any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\n\nThe Corresponding Author also warrants and represents that: (i) they have the full power to enter into this Publication Agreement on their own behalf and on behalf of each Co-Author; and (ii) they have the necessary rights and/or title in and to the Chapter to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licenses expressed to be granted in this Publication Agreement. If the Chapter was prepared jointly by the Corresponding Author and any Co-Author, the Corresponding Author warrants and represents that: (i) each Co-Author agrees to the submission, license and publication of the Chapter on the terms of this Publication Agreement; and (ii) they have the authority to enter into this Publication Agreement on behalf of and bind each Co-Author. The Corresponding Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each such Co-Author.
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\n\n4.2 Nothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\n\n5. TERMINATION
\n\n5.1 IntechOpen has a right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Corresponding Author or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Corresponding Author or any Co-Author (being an individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Corresponding Author or any Co-Author (being a company) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for or enters into any compromise or arrangement with any of its creditors.
\n\nIn case of termination, IntechOpen will notify the Corresponding Author, in writing, of the decision.
\n\n6. INTECHOPEN’S DUTIES AND RIGHTS
\n\n6.1 Unless prevented from doing so by events outside its reasonable control, IntechOpen, in its discretion, agrees to publish the Chapter attributing it to the Corresponding Author and any Co-Author.
\n\n6.2 IntechOpen has the right to use the Corresponding Author’s and any Co-Author’s names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Chapter and has the right to contact the Corresponding Author and any Co-Author until the Chapter is publicly available on any platform owned and/or operated by IntechOpen.
\n\n6.3 IntechOpen is granted the authority to enforce the rights from this Publication Agreement, on behalf of the Corresponding Author and any Co-Author, against third parties (for example in cases of plagiarism or copyright infringements). In respect of any such infringement or suspected infringement of the copyright in the Chapter, IntechOpen shall have absolute discretion in addressing any such infringement which is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\n7. MISCELLANEOUS
\n\n7.1 Further Assurance: The Corresponding Author shall and will ensure that any relevant third party (including any Co-Author) shall, execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\n7.2 Third Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\n7.3 Entire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces and extinguishes all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by or on behalf of the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (together "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of its pre-contract fraudulent misrepresentation or fraudulent concealment.
\n\n7.4 Waiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\n7.5 Variation: No variation of this Publication Agreement shall be effective unless it is in writing and signed by the parties (or their duly authorized representatives).
\n\n7.6 Severance: If any provision or part-provision of this Publication Agreement is or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted.
\n\nAny modification to or deletion of a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\n7.7 No partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Corresponding Author or any Co-Author, nor authorize any party to make or enter into any commitments for or on behalf of any other party.
\n\n7.8 Governing law: This Publication Agreement and any dispute or claim (including non-contractual disputes or claims) arising out of or in connection with it or its subject matter or formation shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of or in connection with this Publication Agreement (including any non-contractual disputes or claims).
\n\nLast updated: 2020-11-27
\n\n\n\n
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