**Abstract**

Reactive oxygen species (ROS) have been associated with a wide variety of human diseases and disorders. The ability of these molecules can incapacitate antioxidant activity leading to an imbalance between oxidants and anti-oxidants, with the latter being more pronounced. ROS are no strangers to immune cell relationships and function and consequently the development of autoimmune and inflammatory diseases. The collateral damage of excessive ROS (collectively called Oxidative stress) to the cells or tissue due to nucleic acid damage and oxidation of macromolecules such as proteins and lipids is linked to the manifestation, malfunction and translation to the disease state of cells. Contrary to this view, recent studies have shown that ROS have protective roles in certain autoimmune and inflammatory diseases. Despite significant advances in our understanding of inflammatory and autoimmune diseases, therapeutics for these diseases still need further development and identification of new targets for improved therapeutic effect. ROS molecules and inflammation modulators appear before disease development making them great therapeutic targets with the potential to inhibit disease manifestation.

**Keywords:** reactive oxygen species, antioxidants, immune cell, autoimmune, inflammatory disease, oxidative stress


**6. Oxidized low-density lipoproteins**

### **6.1. Pathogenesis of oxidized LDL**

In human organism, LDL particles undergo a series of oxidation processes, resulting in reactive oxygen species (ROS) and oxidized LDL (Ox-LDL) particles. These products create negative electric charges that will cause macrophagic stimulation and inflammation.

dysfunction by increasing endothelial adhesivity, by recruiting inflammatory cells into the

Biomarkers in Metabolic Syndrome

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http://dx.doi.org/10.5772/intechopen.79427

Various studies have shown that Ox-LDL levels are associated with MetS. Holvoet et al. demonstrated that patients with MetS had higher Ox-LDL values. They also reported that hyperinsulinemia and impaired glycemic control were associated with increased Ox-LDL levels, independent from lipid levels. The same research found that elevated Ox-LDL levels could

Hurtado-Roca et al. in a study conducted on 3987 subjects demonstrated that Ox-LDL levels are positively correlated with MetS and its components even after adjustments for central obesity and insulin resistance. The strongest association was with triglyceride levels [27].

Pentraxins are a cluster of seric proteins with similar structures and calcium-dependent ligands that play important roles in body protection and in inflammatory mediation. The main mechanism is based on complement activation and interaction with Fc receptors [70].

PTX3 is being produced by immune cells as a response to bacterial substances, endotoxins, IL-1, and TNF-alpha. PTX3 is an acute phase protein with very low serum levels. PTX3 levels rise rapidly as a response to diverse inflammation stimuli. Therefore, PTX3 is considered to be

Recently, it has been shown that increased PTX3 levels are associated with MetS development and progression. In a study conducted on adolescent subjects with obesity, Kardas et al. have shown that subjects with obesity and MetS had higher values of PTX3 than the subjects without MetS. They also observed that low HDL cholesterol and high triglyceride levels were associated with increased PTX3 levels [29]. Also, Zanetti et al. demonstrated that PTX3 was higher in patients with MetS and subclinical atherosclerosis and that PTX3 was independently correlated with low HDL cholesterol levels [30]. Furthermore, a recent study found that PTX3 correlates with the severity of MetS, more precisely, after multivariate analysis PTX3 correlation persisted for glucose level (β = 0.23, p < 0.001), waist circumference (β = 0.37, p < 0.001), and HDL cholesterol (β = −0.31, p < 001) [31]. In conclusion, PTX3 could be a valu-

able biomarker in the prediction of MetS, but further studies should be conducted.

a marker of local and general inflammatory and immune response [71–73].

endothelial wall, and by reducing nitric oxide production **(Figure 6)** [68, 69].

**6.3. Oxidized LDL and metabolic syndrome**

predict the development of MetS in future [26].

**7.1. The role of pentraxins in human organism**

**7.2. Pentraxin-3 and metabolic syndrome**

**7. Pentraxin-3**

During LDL oxidation process, a series of products are generated: fatty acid oxidation products, lipid-derived products, protein oxidation products **(Figure 5)** [63].

Lara-Guzman et al. have shown that THP-1 human macrophage exposure to Ox-LDL caused a series of changes, such as an increased intake of Ox-LDL, overexpression of its receptors, and ROS production. Likewise, in the same study, it has been demonstrated that Ox-LDL determines the synthesis of isoprostanes as oxidation markers and of prostaglandines and prostaglandine metabolites as inflammation markers. Therefore, this study emphasizes that Ox-LDL links oxidative stress with inflammation via macrophages, resulting in systemic and local consequences [64]. Besides that, Schwarz et al. demonstrated that Ox-LDL increases Jun activation domain-binding protein-1 and stimulates inflammatory signaling in macrophages [65].

### **6.2. Oxidized LDL and endothelial dysfunction**

Atherosclerosis represents one of the main alterations caused by MetS, and endothelial dysfunction is the earliest event within it. As mentioned earlier, Ox-LDL triggers inflammation and oxidation process that determines macrophagic activation and ROS production with cytotoxic effect on vascular endothelium [66].

Ox-LDL interacts with lectin-type oxidized LDL receptor 1 (LOX-1) from the surface of endothelial cells and determines their activation [67]. Withal, Ox-LDL causes endothelial

**Figure 6.** LDL oxidation products.

dysfunction by increasing endothelial adhesivity, by recruiting inflammatory cells into the endothelial wall, and by reducing nitric oxide production **(Figure 6)** [68, 69].

## **6.3. Oxidized LDL and metabolic syndrome**

Various studies have shown that Ox-LDL levels are associated with MetS. Holvoet et al. demonstrated that patients with MetS had higher Ox-LDL values. They also reported that hyperinsulinemia and impaired glycemic control were associated with increased Ox-LDL levels, independent from lipid levels. The same research found that elevated Ox-LDL levels could predict the development of MetS in future [26].

Hurtado-Roca et al. in a study conducted on 3987 subjects demonstrated that Ox-LDL levels are positively correlated with MetS and its components even after adjustments for central obesity and insulin resistance. The strongest association was with triglyceride levels [27].


*Article* **Theoretical Performance Evaluation of a Marine Solid Propellant Water-Breathing Ramjet Propulsor**

#### **Nachum E. Eisen \* and Alon Gany**

Faculty of Aerospace Engineering, Technion—Israel institute of Technology, Haifa 3200003, Israel<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>
Received: 24 November 2019; Accepted: 18 December 2019; Published: 20 December 2019

**Abstract:** This work analyzes and presents theoretical performance of a marine water-breathing ramjet propulsor. A conceptual scheme of the motor is shown, the equation of thrust is presented, and the dependence on cruise velocity and depth are discussed. Different propellant compositions, representing a wide variety of formulations suitable for propelling a water-breathing ramjet, are investigated. The theoretical results reveal that the specific impulse of a water-breathing ramjet can increase by as much as 30% compared to a standard rocket, when using a conventional hydroxyl terminated polybutadiene (HTPB)-ammonium perchlorate (AP) propellant, which does not react chemically with the water. When employing a water-reactive propellant containing metal particles such as magnesium or aluminum, the specific impulse may be more than doubled. The thrust coefficient of the propulsor was computed at different cruise velocities and depths and was found to be greater than the predictable drag even at significant depth.

**Keywords:** underwater propulsion; water-breathing ramjet; marine ramjet; water ducted rocket

#### **1. Introduction**

The search for underwater high-speed cruising reveals certain high-thrust marine propulsion concepts which are parallel in principle to their aeronautical counterparts [1]. Rocket engines can propel high-speed underwater vehicles just like aeronautical vehicles. Independent of speed and ambience medium, rocket engines provide very high thrust and do not need air for their operation, hence, they can suite underwater vehicles. However, their specific fuel (propellant) consumption is very high and, thus, they enable only a limited range. Therefore, propulsion principles utilizing the surrounding water as a working fluid as well as a chemical reactant (usually oxidizer), have been considered [2–8]. One of the interesting concepts for propelling high-speed underwater vehicles is the solid propellant water-breathing ramjet [4–8]. It is a powerful propulsion means, independent of atmospheric air, hence, it can operate fully and deeply underwater.

The basic principle of the marine water-breathing ramjet is to utilize the high dynamic (ram) pressure, resulting from the high-speed motion, in order to ingest water into the combustion chamber of the motor, thus increasing the thrust and energetic performance of the propulsor. This work focuses on theoretical prediction of the performance of a water-breathing underwater solid propellant ramjet motor.

The design of an underwater solid propellant water-breathing ramjet is similar to the design of an aeronautical ducted rocket. Water enters the motor through an inlet located in the front of the vehicle and is sprayed through a porous wall into the combustion chamber. Due to the high temperature in the combustion chamber, the incoming water boils and then mixes with the combustion products, forming a high-speed exhaust jet that provides the thrust necessary to propel the vehicle. A conceptual illustration of a solid propellant water-breathing ramjet is presented in Figure 1.

**Figure 1.** Conceptual illustration of a marine solid propellant water-breathing ramjet.

Similarly to solid rocket motors (SRMs), the solid propellant grain can provide high thrust without the addition of water, as well. Nevertheless, the mere addition of water, entering through an inlet due to the vehicle motion, and its further conversion into steam because of the high heat release of the burning solid propellant, increases the thrust and the specific impulse, even with no chemical interaction. Greater performance improvement can be obtained when employing water-reactive propellant compositions, which utilize the incoming water as an oxidizer to increase heat release in addition to using it as a source for steam which increases the mass flow rate of the exhaust jet. Certain metals are known to react very exothermically with water and may be considered for inclusion as water reactive ingredients in propellants. Typically, the reaction of metals with water generates hydrogen besides metal oxides. Table 1 presents theoretical physical and thermochemical properties of selected metals. While the gravimetric heat of reaction is related to the mass of fuel (propellant) carried on board, the volumetric heat of reaction reflects the energy per unit volume and is mainly significant in volume-limited systems.

**Table 1.** Maximum theoretical gravimetric and volumetric heat of reaction and hydrogen generation for selected metals with liquid water, listed in the order of their volumetric heat of reaction (data adapted from [9]).

It is apparent from Table 1 that beryllium reacts extremely exothermically with water both in terms of weight and volume, yet, beryllium and its products are notoriously toxic and therefore are ruled out. Second to beryllium is boron, which demonstrates a promising theoretical heat and gas (i.e., hydrogen) release. In practice, it is hard to obtain high combustion efficiency of boron (though Rosenband et al. [10] have demonstrated combustion of boron in steam), and in addition boron is a relatively expensive ingredient. Hence, for practical applications, aluminum is commonly considered. Aluminum reacts with water to form aluminum oxide (alumina) or aluminum hydroxide, and hydrogen, while releasing a significant amount of heat, as presented in Equation (2). However, aluminum does not combust readily as a thin layer of aluminum oxide naturally forms on the exposed surfaces of the aluminum particles and passivates the metal substrate to inhibit oxidation progression. Different approaches for overcoming the alumina coating problem are discussed extensively in the literature [11–14]. Zirconium has high volumetric heat of reaction, yet lower than that of aluminum. In addition, its other energetic parameter, gravimetric heat of reaction, is substantially inferior to that of

more regularly used metals such as aluminum and magnesium. Relatively to aluminum, magnesium has a lower energy density, but the magnesium-water reaction is easier to initiate. Therefore, some researchers suggested using magnesium [4,5,7,8] or using an aluminum-magnesium mixture [6]. The high-temperature stoichiometric reactions of boron, aluminum, and magnesium with water are presented here:

$$2B + 3H\_2O \rightarrow B\_2O\_3 + 3H\_2 + 397 \frac{kJ}{mol'} \tag{1}$$

$$2Al + 3H\_2O \rightarrow Al\_2O\_3 + 3H\_2 + 818 \frac{kJ}{mol'} \tag{2}$$

$$Mg + H\_2O \rightarrow MgO + H\_2 + 312 \frac{kJ}{mol}.\tag{3}$$

One may note that lithium has a very high gravimetric heat of reaction in addition of its high reactivity with water. However, besides the difficulty in handling lithium, it has been considered for application only as a liquid fuel in its molten phase and not as solid fuel [15].

The chamber pressure in the marine water-breathing ramjet is dictated by the cruise speed, and its maximum value at a given speed is equal to the stagnation pressure of the incoming water flow:

$$P\_0 = P\_a + \frac{1}{2} \rho\_w u^2 \tag{4}$$

where, *P*0 = stagnation pressure; *Pa* = ambient pressure; ρ*w* = water density; *u* = cruise velocity.

The stagnation pressure increases with the cruise speed (as shown in Figure 2); the increased chamber pressure at higher velocities enable enhanced conversion of thermal energy to kinetic energy in the exhaust nozzle, increasing the energetic performance of the motor.

**Figure 2.** Stagnation pressure vs. cruise speed in water (assuming: ρ*w* = 1000 *kg*/*m*3).

#### **2. Theoretical Model**

#### *2.1. Governing Equations and Evaluating Performance*

In order to evaluate the performance of a marine solid propellant water-breathing ramjet, the governing equations are presented. The general thrust equation of a jet engine is:

$$F = \dot{m}\_{\varepsilon}u\_{\varepsilon} - \dot{m}\_{w}u + \left(P\_{\varepsilon} - P\_{a}\right) \tag{5}$$

where, *F* = thrust; . *me* = exhaust mass flow rate; *ue* = exhaust jet velocity; . *mw* = incoming water mass flow rate; *Pe* = exhaust jet static pressure.

The specific impulse (*Isp*) of a propulsion system is an important index defined as the thrust divided by the weight flow rate of propellant consumed in order to produce the thrust, (as shown in Equation (6)). The density specific impulse (ρ*Isp*) of a propulsion system is defined as the specific impulse multiplied by the density of the propellant. For volume-limited vehicles (such as many underwater marine vehicles), the density specific impulse may be of more significant since it represents the thrust produced per volume flow rate of propellant. Thus, the specific impulse and density specific impulse can be used to compare and grade the performance of different propulsion methods. Assuming the nozzle is adapted so that the exhaust pressure is equal to ambient pressure (i.e., optimal expansion ratio for maximal thrust), the specific impulse of a marine ramjet can be calculated as following:

$$I\_{sp} = \frac{F}{\dot{m}\_p g\_0} = \frac{\dot{m}\_c u\_c - \dot{m}\_w u}{\dot{m}\_p g\_0} \, , \tag{6}$$

where, *Isp* = specific impulse; . *mp* = propellant mass flow rate; and *g*0 = universal gravity constant.

The exit (jet) flow rate is equal to the sum of the propellant combustion products and water flow rates, thus: .

$$
\dot{m}\_e = \dot{m}\_w + \dot{m}\_p.\tag{7}
$$

Defining:

$$\frac{w}{p} = \frac{\dot{m}\_w}{\dot{m}\_p} \,'\tag{8}$$

one obtains:

$$I\_{sp} = \frac{\left[1 + \left(\frac{W}{p}\right)\right]\mu\_c - \left(\frac{W}{p}\right)\mu}{\mathcal{S}^0}.\tag{9}$$

Assuming that the combustion chamber pressure is equal to its maximal theoretical value (i.e., the stagnation pressure) and that the ambient pressure is 1 bar (i.e., cruising at sea level), the specific impulse of a marine solid propellant water-breathing ramjet was calculated. The calculations were done according to Equation (9) with the aid of the Computer Program for Calculation of Complex Chemical Equilibrium Compositions and Applications (CEA) [16], which yields the equilibrium flow conditions and jet performance (adapted nozzle). The specific impulse and combustion chamber temperature were calculated for different propellant compositions and with different water/propellant mass ratios.

In this work, a number of propellant compositions were examined. The first composition was a common solid propellant formulation of 85% ammonium perchlorate (AP) and 15% hydroxyl terminated polybutadiene (HTPB), for which the water ingested into the motor undergoes only a phase change into steam with no chemical interaction with the burning propellant. In addition to the non-hydro-reactive AP-HTPB composition, this work deals with certain water-reactive fuels and compositions, representing the potential range of propellants and hydro-reactive propellant ingredients suitable for propelling a marine water-breathing ramjet. One hydro-reactive fuel is pure aluminum, presenting the maximum theoretical performance of aluminized propellants, since it reacts solely with the incoming water with no oxidizer stored onboard. Additional elements examined as potential fuels or propellant ingredients, were boron because of its high heat of reaction (although its practical application may be questionable), and magnesium due to its better reactivity relatively to aluminum and boron. At last, a more practical solid propellant composition, i.e., 70% Mg and 30% polytetrafluoroethylene (PTFE, Teflon), was examined. The small amount of PTFE oxidizer enables initial combustion to take place without the addition of water, but being a fuel-rich propellant, the excessive fuel can react with incoming water.

#### *2.2. Thrust Coe*ffi*cient and Influence of Cruise Depth and Velocity on Performance*

In addition to examining the specific impulse obtained by different propellant compositions at different water to propellant mass ratios, the thrust coefficient and the influence of cruise depth and velocity on performance have been studied. It should be mentioned, that in contrast to the specific impulse which highly depends on the propellant composition and water/propellant ratio, the thrust, and therefore the thrust coefficient, weakly depends on these parameters. Thus, it was chosen in this work to present only the thrust coefficient of the more practical fuel-rich hydro-reactive propellant composition containing 70% Mg and 30% PTFE, with a constant water/propellant ratio of 3.5 (where maximal specific impulse is achieved). Yet, similar results and trends of the thrust coefficient have been observed with other compositions and at different water to propellant ratios.

Assuming the marine water-breathing ramjet is used to propel a cylindrical torpedo-shaped vehicle, the cross-section area of the body may be used as the reference area in computing the thrust and drag coefficients (Equations (10) and (11)). In addition, it was assumed that the nozzle throat diameter is 50% of the body's external diameter.

$$C\_T = \frac{F}{\frac{1}{2}A\rho\_w u^2},\tag{10}$$

where, *CT* = thrust coefficient; *A* = reference area.

It should be mentioned that for a cylindrical torpedo-shaped vehicle with a length to diameter ratio of 15, the drag coefficient is presumed to be smaller than 0.25 at all cruise velocities [17] and may be significantly smaller in a supercavitating vehicle cruising at a high velocity [18]. In order for the vehicle to overcome the drag force, the thrust coefficient has to be equal or larger than the drag coefficient, which is related to the same reference area:

$$\mathcal{C}\_D = \frac{D}{\frac{1}{2}A\rho\_{w}u^2},\tag{11}$$

where, *CD* = drag coefficient; *D* = drag force.

Assuming isentropic supersonic flow in the divergent section of the nozzle, the data obtained from the thermochemical code, mentioned above, enable to calculate the exit velocity and pressure as presented in Sutton and Biblatz [19], and thus the thrust (Equation (5)).

The expansion ratio is defined as the ratio between the nozzle's exit and throat cross-section areas. The results presented in this study refer to a ramjet with an expansion ratio of two cruising at a velocity in the range of 50 to 150 m/s at a depth of 0 to 100 m below sea level. Atmospheric pressure (1 bar) is assumed at sea level and a pressure increase by 1 bar every 10 m below sea level.

#### **3. Results and Discussion**

#### *3.1. Performance of Di*ff*erent Propellant Compositions*

#### 3.1.1. Non-Hydro-Reactive Composition

Figure 3a presents the calculated specific impulse and density specific impulse for a solid propellant water-breathing ramjet motor with a propellant grain consisting of 85% AP and 15% HTPB versus the water to propellant (w/p) mass ratio for two cruise speeds, 25 m/s (implying chamber pressure of 4 bar) and 100 m/s (implying chamber pressure of 50 bar). The values at a zero w/p ratio represent the specific impulse of a pure rocket (no water addition). In general, the w/p ratio is limited by the decrease of the overall chamber temperature to the water equilibrium boiling (condensation) temperature (Figure 3b). It is obvious that the addition of water increases the specific impulse (and thus the density specific impulse) substantially. However, at the low cruise speed (low chamber pressure), the specific impulse is inferior to that of a rocket motor operating at standard conditions (chamber pressure of about 69 bar). Nevertheless, the performance of the water-breathing ramjet at the higher chamber pressure (50 bar, corresponding to a cruise speed of 100 m/s) is noticeably higher than that of the lower pressure due to the contribution of the greater expansion in the nozzle. When cruising at 100 m/s, the specific impulse exceeds that of a standard rocket motor by as much as 30% when adding water at a w/p ratio of 2.5.

**Figure 3.** (**a**) Calculated specific impulse—*Isp*, density specific impulse—ρ*Isp*, and (**b**) combustion chamber temperature—*Tc*, of an underwater water-breathing ramjet using a non-hydro-reactive solid propellant (85% ammonium perchlorate (AP) + 15% hydroxyl terminated polybutadiene (HTPB)) vs. water/propellant mass ratio, for two different cruise velocities.

#### 3.1.2. Hydro-Reactive Fuels

Figure 4a presents the theoretical specific impulse and density specific impulse for a water-breathing motor with aluminum as a fuel versus the water to propellant (fuel) mass ratio, for two cruise speeds, 25 and 100 m/s, corresponding to low (4 bar) and high (50 bar) chamber pressures, respectively. In this case, even at the low speed cruise conditions, the theoretical specific impulse of the water-breathing ramjet exceeds that of a standard rocket, the density specific impulse exceeds that of a standard rocket even more so due to the relatively high density of aluminum (compared to HTPB-AP based propellants). Figure 4b presents the calculated combustion chamber temperature versus water/propellant mass ratio. It can be observed that at low w/p ratios (bellow the stoichiometric ratio) the addition of water increases the chamber temperature due to the increase in available oxidizer that enables more aluminum to react and release heat. On the other hand, at large w/p ratios, the addition of water reduces the temperature, similarly to the trend shown above in the case of a non-hydro- reactive propellant.

To be more realistic, one can take into account the fact that the reaction products of aluminum with water contain large amounts of condensed material (Al2O3 or Al(OH)3) that may be retained within the motor. Hence, calculations were repeated for the case where the condensed material remains in the reaction chamber and does not exit the nozzle. Even in such case, the motor performance may be doubled and more, compared to standard solid rockets.

**Figure 4.** (**a**) Calculated specific impulse—*Isp*, density specific impulse—ρ*Isp*, and (**b**) combustion chamber temperature—*Tc*, of an underwater water-breathing ramjet using aluminum as a hydro-reactive fuel vs. water/propellant mass ratio, for two different cruise velocities.

As mentioned above, theoretically, boron as a fuel may provide the uppermost performance of a water-breathing ramjet propulsor (assuming beryllium is ruled out due to toxicity), though there is uncertainty regarding practical application. Additionally, magnesium is widely considered as a fuel for a marine ramjet due to its reactivity. Figures 5a and 6a present the theoretical specific impulse and density specific impulse of a ramjet using boron and magnesium as fuels at two cruise speeds (100 m/s and 25 m/s). It is apparent from Figure 5a that at a cruise speed of 100 m/s boron enables to achieve superior theoretical performance of up to four times the specific impulse achieved by an SRM, yet, even at 25 m/s a significant specific impulse may be achievable. It should be noted, that due to the immense amount of heat released from the boron-water reaction, maximal specific impulse is achieved at a very large w/p ratio. Figure 6a reveals that the specific impulse of magnesium is inferior to that of aluminum by roughly 15% at both cruise speeds. However, due to magnesium's reactivity, which possibly implies increased combustion efficiency, magnesium is a promising fuel and can be used as an energetic propellant ingredient to improve performance of solid propellant marine ramjets. Figures 5b and 6b present the combustion chamber temperature of a ramjet using boron and magnesium as fuels, respectively. The trends discussed above in the case of aluminum, of an increase in temperature at a low w/p ratio and a decrease in temperature at a large w/p ratio, can be observed also in the cases of boron and magnesium. It should be commented, that while aluminum (Figure 4b) and magnesium (Figure 6b) present roughly an equal peak temperature of 3100 K, the peak temperature obtained with boron (Figure 5b) is significantly lower due to the large latent heat and the relatively low temperature of evaporation of the water-boron reaction product, boron oxide. In all cases, where phase change occurs (observed as "plateaus" in the temperature figures), the phase change temperature is greater for the higher velocity due to the increased chamber pressure, which results in an elevated equilibrium vapor pressure.

**Figure 5.** (**a**) Calculated specific impulse—*Isp*, density specific impulse—ρ*Isp*, and (**b**) combustion chamber temperature—*Tc*, of an underwater water-breathing ramjet using boron as a hydro-reactive fuel vs. water/propellant mass, for two different cruise velocities.

**Figure 6.** (**a**) Calculated specific impulse—*Isp*, density specific impulse—ρ*Isp*, and (**b**) combustion chamber temperature—*Tc*, of an underwater water-breathing ramjet using magnesium as a hydro-reactive fuel vs. water/propellant mass ratio, for two different cruise velocities.

#### 3.1.3. Practical Hydro-Reactive Composition

In practice, water-reactive ingredients may be embedded within a fuel-rich propellant matrix containing a small fraction of oxidizer. Figure 7a presents the calculated specific impulse and density specific impulse for a water-breathing ramjet motor with a propellant grain consisting of 70% Mg and 30% PTFE versus the water to propellant mass ratio for two cruise speeds. This propellant composition is fuel-rich, thus also in this case maximum temperature is achieved when water is introduced in the combustion chamber as an additional oxidizer reacting with the excess magnesium, as can be seen in Figure 7b. It is apparent from Figure 7a, that the addition of a hydro-reactive fuel ingredient such as magnesium, increases significantly the performance relatively to the case of a non-hydro-reactive composition presented at the beginning. At a cruise speed of 100 m/s it enables one to achieve a specific impulse double that achieved with a standard solid propellant rocket.

**Figure 7.** (**a**) Calculated specific impulse—*Isp*, density specific impulse—ρ*Isp*, and (**b**) combustion chamber temperature—*Tc*, of an underwater water-breathing ramjet using a hydro-reactive fuel-rich solid propellant (70% Mg + 30% PTFE) vs. water/propellant mass ratio, for two different cruise velocities.

#### *3.2. Thrust Coe*ffi*cient and Influence of Cruise Depth and Velocity on Performance*

Figure 8 presents the thrust coefficient and the specific impulse of a marine water-breathing ramjet propelled by a solid propellant grain containing 70% magnesium and 30% PTFE with a w/p ratio of 4 versus the cruise speed at different depths. It is apparent from Figure 8a,b that with the increase in depth, the thrust coefficient and the specific impulse decrease. The reason is that the increase in ambient pressure reduces the exit nozzle performance (see Equation (5)). Yet, even at a depth of 100 m below sea level, the thrust coefficient is greater than the drag coefficient predicted on a torpedo-like vehicle discussed above, thus the motor can suite a broad range of velocities and depths. If necessary, the selection of a different throat diameter will enable to achieve a lower or higher thrust level (that will dictate a different propellant consumption rate).

**Figure 8.** Calculated (**a**) thrust coefficient and (**b**) specific impulse of an underwater water-breathing ramjet with a nozzle expansion ratio of two and a throat diameter 50% of the vehicle's external diameter, using a hydro-reactive fuel-rich solid propellant (70% Mg + 30% PTFE) with a water/propellant mass ratio of 3.5 vs. cruise velocity, cruising at a depths of 0, 50, and 100 m below sea level.

#### **4. Conclusions**

The concept of a water-breathing ramjet for underwater propulsion has been evaluated theoretically based on thermochemical calculations and available practical propellants and energetic components. It is revealed that theoretically, even without chemical interaction, a solid propellant ramjet motor augmented by water entering from the surroundings through an inlet, can deliver specific impulse higher by 30% than a standard solid rocket. By introducing a fuel-rich propellant containing large amounts of a water-reactive metal such as magnesium, aluminum, or boron, the specific impulse may be doubled or more. At cruise speeds as low as 25 m/s, the performance of a water-breathing ramjet with a non-water-reactive propellant is inferior to that of a standard rocket, however, when the propellant is enriched with water-reactive ingredients, improvement in performance can be achieved even at relatively low velocities. In all cases, the specific impulse increases significantly with speed because of the increase in chamber pressure. At sea level, the thrust coefficient is almost constant at velocities between 50–150 m/s, whereas at greater depths there is a decrease in both the thrust coefficient and the specific impulse. Yet, at high velocities the thrust coefficient decreases mildly with depth, thus, the marine solid propellant water-breathing ramjet is a powerful propulsion method suitable for propelling high-speed underwater vehicles at a wide range of depths.

**Author Contributions:** Conceptualization, N.E.E. and A.G.; Data curation, N.E.E.; Formal analysis, N.E.E. and A.G.; Funding acquisition, A.G.; Investigation, N.E.E.; Methodology, N.E.E. and A.G.; Project administration, A.G.; Resources, A.G.; Software, N.E.E.; Supervision, A.G.; Validation, A.G.; Visualization, N.E.E.; Writing—original draft, N.E.E.; Writing—review & editing, A.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by PMRI—Peter Munk Research Institute—Technion.

**Acknowledgments:** The authors wish to thank David Albagli for his advice with the thermochemical calculations.

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

#### **References**

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


*Article* **Calcination Enhances the Aflatoxin and Zearalenone Binding E**ffi**ciency of a Tunisian Clay**

**Roua Rejeb 1,2,\*, Gunther Antonissen 2,3, Marthe De Boevre 4, Christ'l Detavernier 4, Mario Van de Velde 4, Sarah De Saeger 4, Richard Ducatelle 2, Madiha Hadj Ayed 1 and Achraf Ghorbal 5**

Received: 3 September 2019; Accepted: 14 October 2019; Published: 16 October 2019

**Abstract:** Clays are known to have promising adsorbing characteristics, and are used as feed additives to overcome the negative effects of mycotoxicosis in livestock farming. Modification of clay minerals by heat treatment, also called calcination, can alter their adsorption characteristics. Little information, however, is available on the effect of calcination with respect to mycotoxin binding. The purpose of this study was to characterize a Tunisian clay before and after calcination (at 550 ◦C), and to investigate the effectiveness of the thermal treatment of this clay on its aflatoxin B1 (AFB1), G1 (AFG1), B2 (AFB2), G2 (AFG2), and zearalenone (ZEN) adsorption capacity. Firstly, the purified clay (CP) and calcined clay (CC) were characterized with X-ray Fluorescence (XRF), X-ray Diffraction (XRD), Fourier transform infrared spectroscopy (FTIR-IR), cation exchange capacity (CEC), specific surface area (SBET), and point of zero charge (pHPZC) measurements. Secondly, an in vitro model that simulated the pH conditions of the monogastric gastrointestinal tract was used to evaluate the binding efficiency of the tested clays when artificially mixed with aflatoxins and zearalenone. The tested clay consisted mainly of smectite and illite. Purified and calcined clay had similar chemical compositions. After heat treatment, however, some changes in the mineralogical and textural properties were observed. The calcination decreased the cation exchange capacity and the specific surface, whereas the pore size was increased. Both purified and calcined clay had a binding efficacy of over 90% for AFB1 under simulated poultry GI tract conditions. Heat treatment of the clay increased the adsorption of AFB2, AFG1, and AFG2 related to the increase in pore size of the clay by the calcination process. ZEN adsorption also increased by calcination, albeit to a more stable level at pH 3 rather than at pH 7. In conclusion, calcination of clay minerals enhanced the adsorption of aflatoxins and mostly of AFG1 and AFG2 at neutral pH of the gastrointestinal tract, and thus are associated with protection against the toxic effects of aflatoxins.

**Keywords:** aflatoxins; zearalenone; clay; purified; calcined; adsorption; pH

**Key Contribution:** In vitro binding capacity of purified and calcined clay was determined for AFB1, AFB2, AFG1, AFG2 and ZEN in the pH of the poults gastrointestinal tract. The calcined clay has successfully improved the binding efficiency of AFB2, AFG1, AFG2 and ZEN.

#### **1. Introduction**

Mycotoxins are secondary metabolites produced by toxigenic fungi growing on a wide range of agricultural products [1]. The Food and Agriculture Organization (FAO) of the United Nations stated that nearly 25% of the cereal products is contaminated with mycotoxins [2].

Aflatoxins (AFs) are considered as the most important mycotoxins in food and feed because of their carcinogenicity (IARC monograph class I) and their high prevalence, especially in Southern regions [3,4]. AFs are a group of heterocyclic metabolites that are mainly produced by members of the genus *Aspergillus*, contaminating agricultural commodities. *Aspergillus* fungi are both found in the field as storage pathogens, therefore the AFs' content is omnipresent, but with a tendency to increase during storage [5,6]. AFs cause serious health problems, economic losses, and deleterious effects on performance in a variety of farm animals including pigs, poultry, and cattle [7]. More specifically in poultry, AFs reduce the growth rate, decrease egg production, induce changes in organ weight, and increase the risk of disease [4]. In young chicks, AFs reduce immune competence and cause liver damage [8–10]. Aflatoxin B1 (AFB1) is the most potent of the aflatoxins, followed by AFG1, AFB2, and AFG2 [11]. AFB1 comprise a greater potency associated with the cyclopentenone ring of the B series, when compared with the six-membered lactone ring of the G series [12].

Zearalenone (ZEN) is a phenolic resorcyclic acid lactone mycotoxin produced by *Fusarium* fungi growing on cereal grains and derived products worldwide. *Fusarium* mycotoxins are primarily produced before harvest, on the field. ZEN is a potent estrogenic metabolite as it has the ability to bind to estrogen receptors and induces estrogenic alterations including uterine enlargement, swelling of the vulva and mammary glands, and pseudopregnancy [13,14].

To offset the negative effects of mycotoxins on animal health, a wide range of mycotoxin decontamination strategies has been reported in the literature [15–17]. Adding mycotoxin binders to the feed is probably the most common post-harvest mitigation approach [18–20]. Binders decrease the absorption of mycotoxins from the gastrointestinal (GI) tract into the blood circulation and target organs by adsorbing them on their surface [21], forming a binder-toxin complex which is eliminated through the fecal material [22]. Inclusion of mycotoxin binders improves the average daily gain and the average daily feed intake in pigs and reduced the injurious effect of AFs on body weight, feed conversion ratio (FCR), serum alanine aminotransferase (ALT), and urea concentration in broiler chickens [8,23]. However, it should be noted that adding a high dose of clay in the feed might cause nutrient deficiency by adsorbing micronutrients, vitamins and organic compounds, while also having negative effects on the bioavailability of minerals and trace elements [15,24]. In addition, the risk of the contamination of raw clays with metals and dioxins has to be considered [25]. Among mycotoxin binders, clay minerals are the largest group. Several aluminosilicate clays such as hydrated sodium calcium aluminosilicate (HSCAS), bentonite, montmorillonite, smectite, and zeolite have good binding efficiency to mycotoxins [26,27]. These clays mostly bind small mycotoxins, such as AFs and ochratoxin A, but have less binding affinity for the larger molecules of certain *Fusarium* toxins. ZEN can be adsorbed by only a limited number of binders with a large variation in binding capacity [28]. Effectiveness of binding also depends on the type, and the dosage of the binders. A Brazilian study showed that about 64% of the products on the market were ineffective in binding AFs [29,30].

Clays are used in their natural state or treated through various processes such as calcination, acid activation, pillaring, organic modification with polymers, or cation and anion exchange. These modified clays can be more effective in binding some mycotoxins than the untreated clay [31–34]. Calcination is a process in which clay minerals are heated to different temperatures [33]. It removes the water located in intra-crystalline tunnels, which changes the pore structure and surface proprieties [35,36]. Consequently, heat treatment has an impact on the specific surface area of clay, which is responsible for the adsorption capacity [37]. Calcined clays have been used in numerous studies as adsorbents to eliminate heavy metals and cationic dyes [38,39].

To the best of the authors' knowledge, only one study investigated the effect of calcination on mycotoxin binding [40]. In this paper it was reported that the adsorption of AFB1 was reduced after calcination of a bentonite clay. It is questionable whether this observation can be extrapolated to other types of clay and other mycotoxins. Therefore, the purpose of the present study was to analyze the physical characteristics and to evaluate the binding of different mycotoxins before and after calcination of a montmorillonite type of clay from Tunisia.

#### **2. Results**

#### *2.1. Chemical Characterization*

XRF analyses revealed that purified native clay (CP) and its calcined form (CC) were mainly composed of silicon dioxide (SiO2), aluminum oxide (Al2O3), calcium oxide (CaO) and iron oxide (Fe2O3) (Table 1). In addition, the occurrence of magnesium oxide in both samples can be due to the presence of smectite, and also to a small amount of dolomite, which is confirmed by the results of XRD and FTIR-IR [41].

**Table 1.** Chemical composition of clays.

CP: purified clay, CC: calcined clay.

#### *2.2. Infrared Spectroscopy Characterization (FTIR-ATR)*

The FTIR patterns of the CP (Figure 1) exhibits several characteristic bands corresponding to the stretching vibrations of the surface hydroxyl groups (Si–Si–OH, or Al–Al–OH) at 3695 cm−1 which indicates the presence of kaolinite [40–44]. The spectrum shows a characteristic band of montmorillonite at 3620 cm−1 [43]. The band at 1432 cm−1 corresponds to carbonate [calcite (Ca CO3) or dolomite (Ca, Mg (CO3)2)] [44–46]. The band at 711 cm−1 corresponds to calcite [44,45]. The band at 518 cm−1 is due to Si–O–Al (octahedral) bending vibration [46,47]. Vibration at 1635 cm−1 was attributed to the bending of adsorbed water. The band of deformation near to 873 cm−1 indicates that the clay is octahedral [48], while the band near 910 cm−1 corresponds to an Al-O-H deformation characteristic of dioctahedral smectite [49].

After calcination, the water OH-bending (1635 cm−1) mode totally disappeared. This is a consequence of dehydroxylation and dehydration by the thermal treatment.

**Figure 1.** Infrared spectra of purified clay (CP) and calcinated clay (CC).

#### *2.3. X-Ray Di*ff*raction (XRD)*

XRD characterization as presented in Figure 2 showed that CP and CC consisted mainly of calcic smectite, as demonstrated by the main peak near 14.10 Å and the additional peaks at 4.48 Å and 2.56 Å [50]. The XRD patterns confirmed the presence of kaolinite by the basal spacing at 7.16 Å, 3.84 Å, and 3.57 Å [45,50,51]. The characteristic reflection of dolomite was observed at 2.89 Å [45], while that of calcite was observed at 3.03 Å and 1.90 Å [45,50,52]. Both clays were also characterized by the presence of quartz, indicated by several peaks at 4.25 Å, 3.34 Å, 2.28 Å, 2.09 Å, and 1.87 Å [47,48,53,54]. Following calcination, the characteristic peaks of kaolinite at 7.16 Å and 3.57 Å disappeared, while a new peak appeared at 9.91 Å.

**Figure 2.** X-ray diffractograms of purified and calcined clay (S: smectite, I: illite, K: kaolinite, Q: quartz, C: calcite, D: dolomite), CP: purified clay, CC: calcined clay.

#### *2.4. Cation Exchange Capacity (CEC)*

The thermal treatment resulted in a decreased CEC (9.28 Cmol(+) (kg−1) for CC) and a reduction in Ca, Mg and Na after calcination (Table 2).

**Table 2.** Cation exchange capacity of purified clay (CP) and calcined clay (CC).

CP: purified clay, CC: calcined clay, CEC: cation exchange capacity.

#### *2.5. BET Surface Analysis*

Brauner-Emmett-Teller (BET) N2 adsorption/desorption analysis showed a surface area of CP 64.06 m2/g. Thermal treatment resulted in a decreased surface area: 44.42 m2/g for CC (Table 3) and an increase in pore size from 57.03 Å for CP to 66.72 Å for CC.

**Table 3.** Textural characteristic of clay minerals.

SBET: BET Surface Area, CP: purified clay, CC: calcined clay.

#### *2.6. Point of Zero Charge (PZC)*

As can be seen in Figure 3, the pHPZC of CP was 9.94, while it was 10.03 for the CC.

**Figure 3.** Determination of point of zero charge of purified clay (CP) and calcined clay (CC).

#### *2.7. Mycotoxin Binding E*ffi*ciency*

At pH of 3, CC and CP were able to bind 100% of AFB1. However, compared to CP, CC had a higher (*p* < 0.05) adsorption capacity for AFB2, AFG1 and AFG2 (Table 4). Moreover, CP was not able to bind ZEN at pH 3, while the calcinated clay did adsorb ZEN (*p* < 0.05). Table 5 contains the results of mycotoxin adsorption of the CP and CC at pH 7. The adsorption of AFB1, AFB2, AFG1, and AFG2 at pH 7 was significantly higher (*p* < 0.05) for CC compared to CP. As almost no ZEN was bound to CP, the adsorption of ZEN at pH 7 was significantly higher (*p* < 0.05) for the CC (41 ± 12%) than the CP (1 ± 1%).

**Table 4.** In vitro adsorption of AFB1, AFB2, AFG1, AFG2 and ZEN by purified clay (CP) and calcined clay (CC) at pH 3. Results are presented as mean ± SD. Means of CC indicated with \* are significantly different compared to CP (*p* < *0.05*). (NS: not significantly different).

**Table 5.** In vitro adsorption of AFB1, AFB2, AFG1, AFG2 and ZEN by purified clay (CP) and calcined clay (CC) at pH 7. Results are presented as mean ± SD. Means of CC indicated with \* are significantly different compared to CP (*p* < 0.05).

Based on these in vitro adsorption data, the in vitro binding efficiency of AFs and ZEN of the purified and calcined clay was predicted (Figure 4). The results show that both clays exhibited a strong in vitro binding affinity to AFs. Heat treatment of the clay improved the binding efficiency of ZEN (29 ± 18%) compared to the CP (0 ± 0%). The binding efficiency of AFB1 was 94 ± 3% and 99 ± 0% for the CP and CC, respectively. The effectiveness of CP and CC in binding AFB2 was 86 ± 9% and 99 ± 1%, respectively. The in vitro binding efficiency of AFG1 and AFG2 was significantly higher (*p* < 0.05) for the CC than the CP. AFG1 binding efficiency was 59 ± 18% for CP and 98 ± 2% for CC (*p* < 0.05). Furthermore, the binding efficiency of AFG2 was 15 ± 38% for the CP and 96 ± 2% for the CC. These results demonstrate that the thermal treatment of the clay enhances the binding efficiency of AFG1 and AFG2.

**Figure 4.** In vitro binding efficiency (%) of AFB1 (5 ng/mL), AFB2 (5 ng/mL), AFG1 (5 ng/mL), AFG2 (5 ng/mL) and ZEN (25 ng/mL) of purified clay (CP) and calcined clay (CC). Data are represented as mean values ± SD. Means of CC indicated with \* are significantly different compared to CP (*p* < 0.05)

#### **3. Discussion**

Several reports have shown that adding phyllosilicate clay supplements to animal feed represents one of the most powerful prevention strategies for aflatoxicosis in livestock [21,55–57]. Heat treatment may alter the adsorbing properties of the clays, however, this has not been investigated much with respect to mycotoxins so far.

Our results show that the chemical composition of the studied clay was not affected after heat treatment. Similar results were found in another study with a different type of clay [40]. Indeed, there is no change in bentonite composition before and after calcination. The chemical composition of the studied clay is a typical composition of south-eastern Tunisian clays [45]. CP and CC were also characterized by a high calcium oxide content and a low percentage of sodium oxide which are in accordance with previous work [48].

Disappearance of the characteristic peaks of kaolinite following calcination, as observed by XRD, is probably due to the dehydroxylation of the kaolinite structure, leading to the collapse of clay mineral structure [40,50,58]. Aluminosilicate minerals are characterized by a CEC varying from 10 (meq/100 g) for kaolinite minerals to 100 (meq/100 g) for illite and smectite minerals [59]. The tested clay showed a low value of CEC which can be due to the poor crystallinity of smectite and to the presence of kaolinite, illite, and some impurities (calcite, quartz, dolomite, etc.). The decrease in CEC is due to the dehydration and dehydroxylation of smectite which results in a collapse of the interlayer [53,60]. This result is in agreement with previous reports [34,61] stating that the thermal treatment reduces the cation exchange capacity of clays. The surface area is an important property for clay adsorption capacity. Previous work reported that the surface area decreases with increasing temperatures of thermal treatment [33,46,54]. Calcination of clay at 560 ◦C causes an agglomeration of the particles leading to a 15% decrease in the surface area and an increase in the pore size [62]. According to the literature [35], the increase in the diameter of pores is due to a reduction of the basal spacing through dehydration of the interlayer spaces which is a consequence of a first collapse of smectite layers. Similar research observed that calcination of clay affects the interlayer space [63,64]. The pHPZC is an important characteristic of minerals and usually used to define the state of the surface of a dispersed solid phase at the solid-electrolyte solution surface and may indicate the ionization of functional groups and their possible interaction with the mycotoxin molecules [65]. In addition, low-charge montmorillonites or other low-charge smectites would be more effective feed additives than higher-charge clays [34]. The surface is negative at pH>pHPZC, positive at pH<pHPZC and neutral at pH=pHPZC [66]. The obtained results for pHPZC in our study indicated that the surface of the studied clays is positive at pH3 and pH7 and are moderately higher than the pHPZC reported in the literature for Tunisian smectitec clays (8.2) [67]. The use of adsorbents mixed with food and feed is one of the prominent post-harvest approaches to protect against mycotoxins toxicity, which are supposed to bind efficiently mycotoxins in the gastrointestinal tract. In the current study, we evaluate the binding capacity of AFs and ZEN of purified and calcined clay. The in vitro ZEN and AFs adsorption is assessed at pH 3 and pH 7 which are representative for the GI tract of the monogastric animals. Similar models were successfully applied in previous in vitro experiments [21,59,68]. Regarding adsorption and desorption of ZEN, the adsorption of ZEN by clay is usually lower than aflatoxins [21]. This could be because of the low polarity of ZEN compared to AFs [19]. Furthermore, ZEN has a more spherical molecular geometry than the planar structure of AFs [18]. Moreover, in a recent study [21], it was reported that ZEN is significantly less adsorbed in alkaline than in acid and neutral conditions. De Mil et al. [18] suggested that the pH may influence the phenolic hydroxyl group of ZEN or the ionization-state of the functional groups of the mycotoxin binders, and thereby alter the adsorption by the ionic interactions.

Our findings confirmed a better adsorption capacity for ZEN for the calcined clay than the purified native clay. It has been reported that modified clays have been developed to improve ZEN adsorption, which provide sufficient space between the layers to react with mycotoxin with a relatively less polarity with the appropriate electrical charging [69–72]. These modified surface properties lead to greater hydrophobicity by exchanging the structural load balance cations with high molecular

weight quaternary amines [27]. Feng et al. [73] have concluded that the modified clays have led to low desorption rates, with higher ZEN adsorption than the non-modified clay. The increase in adsorption capacity of AFs and ZEN with calcined clay in our study might be related to the increase in pore size (Table 3), and the decrease in CEC (Table 2) of clay after heat treatment at 550 ◦C. Chen et al. [35] reported that calcination of palygorskite at high temperatures improves dye adsorption efficiency, which is basically associated with a larger size and a wider size distribution of pores. In addition to the pore size, CEC play an important role in the adsorption phenomenon [18]. Exchangeable cations neutralize the interlayer charges in phyllosilicates, and are involved in the binding of AFB1 [7,74]. Deng et al. [75] suggested that layer charge density and the type of exchange cations have a distinct influence on the adsorption of AF. Recent research [43] demonstrated that heating of bentonite improves the clays' adsorption affinity and capacity of AFB1, which is mainly because of the reduction of cation exchange capacity. Furthermore, AFB1 is a polar mycotoxin and contains ß-carbonyl, which is involved in the adsorption process [55]. According to Prapapanpong et al. [21], the adsorption process involves the exchange of electrons of the metallic cation on the surface of the adsorbent, especially the positive charge of calcium ions on each layer of clay. A hypothesis proposed by Jaynes et al. [76] discussed the possibility that aflatoxins can be captured at multiple locations on hydrated sodium calcium aluminosilicate (HSCAS) surfaces, as well as between HSCAS inter-layers.

#### **4. Conclusions**

In this study, calcination improved the adsorption efficacy of the clay for AFB2, AFG1, AFG2, and ZEN. The adsorption-desorption rate showed that the pH condition in the GI tract might influence ZEN adsorption rate. The authors conclude that this specific clay has the potential to be used as a mycotoxin binder in poultry and probably for other animal species.

Unraveling the exact binding-mechanism, including the verification of the kinetics of mycotoxin binders has to this day remained largely uninvestigated. However, these data are of crucial importance to further ameliorate mycotoxin binders' efficacies, and to aim for a wider adaption of these mitigation strategies in both animals and humans.

#### **5. Materials and Methods**

#### *5.1. Chemical Products and Reagents*

Methanol (LC-MS/MS grade) and glacial acetic acid (LC-MS/MS grade were procured from Biosolve B.V. (Valkenswaard, The Netherlands). Acetic acid and ammonium acetate (analytical grade) were supplied by Merck (Darmstadt, Germany). A Milli-Q® SP Reagent water apparatus (Millipore; Brussels, Belgium) was used for water purification. For the mycotoxin standards, AFB1, AFB2, AFG1, AFG2 and ZEN were purchased from Sigma-Aldrich (Bornem, Belgium). Disinfectol® (denaturated ethanol + 5% ether) was supplied by Chem-Lab (Belgium). Mycotoxin standards were dissolved in methanol (1 mg/mL), and were storable for a minimum of 1 year at −18 ◦C. Other chemicals and reagents were of analytical grade.

#### *5.2. Source and Preparation of Clay*

The raw clay sample was collected from Jebel Aïdoudi (El Hamma, Gabes) in the southern part of Tunisia. To purify the clay, the sample was ground and wet-sieved with an electromagnetic sieve shaker (Matest S.p.A., Triviolo, Italy) in order to eliminate all the impurities. Next, they were dried in an oven (Memmert GmbH + Co.KG, Schwabach, Germany) at 105 ◦C. The obtained clay was subsequently washed with distilled water until separation of the liquid/solid phases became difficult, and were dried again in an oven at 105 ◦C. The thermally-treated clay was obtained by a calcination process of the purified clay in a muffle furnace (Sirio Dentel Srl, Meldola, Italy) at 550 ◦C for 5h. Subsequently, purified clay (CP) and calcined clay (CC) samples were ground and sieved into fine powder (≈100 μm).

#### *5.3. Physico-Chemical Characterization of Clays*

#### 5.3.1. X-Ray Fluorescence (XRF)

The chemical composition of the minerals present in the two clays was performed using the XRF spectrometer model Thermo OASIS 9900 (Thermo Fisher Scientific (Schweiz) AG, Bâle, Suisse). It was performed in order to know the elemental oxides that are present in the clays.

#### 5.3.2. X-Ray Diffraction (XRD)

XRD was performed to identify the mineral composition of clays and the crystal line phases. It was conducted by an X-ray diffractometer « PANALYTICAL X'PERT PROMPD » using Cu Kα radiation (λ = 0.154 nm) with the voltage of 45 kV, 40 mA. Then the basal spacing of samples was determined from Bragg's law (*n* λ= 2 d sin θ), where 'n' is the path difference between the reflected waves, equal to an integral number of wavelengths (λ), 'λ' the wavelength (nm), 'd' the interlayer spacing (nm), and 'θ' the diffraction angle (◦).

#### 5.3.3. Fourier Transformed Infrared Spectroscopy (FTIR-ATR)

FTIR-IR helps in the identification of various forms of the minerals present in the clay. The vibrational spectra were performed with FTIR with a Spectrum Two PerkinElmer spectrometer, in the attenuated total reflection (ATR) mode, with a highly sensitive Deuterated Triglycine Sulfate (DTGS) detector. The samples were scanned 10 times in the range of 500–4000 cm−1, with a 2 cm−1 spectral resolution.

#### 5.3.4. Point of Zero Charge (PZC)

The Chemistry surface characterization was performed according to the solid addition method described by Noridine et al. [77] with slight modifications. A series of 50 mL of 0.01 mol/mL of NaCl (0.1 M) solutions were poured in beakers. The pH was adjusted in the range of 2–12 with 0.1 M HCl or 0.1 M NaOH solutions. Then, 0.2 g of clay was soaked in each solution under agitation at room temperature, and the final pH was measured after 24 h. The pHPZC is the point in the curve when the pHinitial (pHi) verses pHfinal (pHf) intersects the line (pHi = pHf).

#### 5.3.5. Cation Exchange Capacity (CEC)

CEC of clays was conducted using the barium chloride method [78]. Briefly, samples between 1 g and 5 g, depending on their probable CEC, were weighed into 50 mL centrifuge tubes. Then, 30 mL of 0.1 M BaCl2 was added, and the tubes were shaken slowly on a reciprocal shaker for 2 h. The samples were then centrifuged at 2500 rpm for 10 min and the supernatant solution were filtered through Whatman 42 filter paper. The solution was collected in polyethylene bottles for analysis.

#### 5.3.6. BET Surface Analysis

The textural properties of the clays (specific surface area, porosity) were determined by nitrogen adsorption using the multipoint Brunauer-Emmet-Teller (BET) method (ASAP 2020 Micromeritics Instruments, Norcross, GA, USA). The specific surface area was measured at 77 K and the pore size distribution was calculated in the radius range from 2000 to 100,000 nm by the BJH method using the adsorption isotherm.

#### *5.4. Mycotoxin Adsorption*

In vitro evaluation of the adsorption capacity of both clays for AFB1, AFB2, AFG1, AFG2, and ZEN was adopted from Di Mavungu et al. [79] with slight modifications. To determine the effect of pH on the mycotoxin binding capacity within the pH range of the gastrointestinal tract of poults, tests were performed at pH 3 and 7. In brief, 50 mg of clay sample was shaken and incubated for 3 h in 10 mL citrate buffer (pH 3) or phosphate buffer (pH 7) together with AFB1, AFB2, AFG1, and AFG2 at a concentration of 5 ng/mL, or with ZEN at a concentration of 25 ng/mL.

The mycotoxins were determined by LC-MS/MS using previously described methods [80]. A Waters Acquity UPLC system coupled to a Quattro Premier XE mass spectrometer (Waters, Milford, MA, USA) equipped with a Z-spray electrospray ionization (ESI) interface was used for the determination and quantification of mycotoxins. Chromatographic separation was achieved using a Symmetry C18 column (5 μm, 150 × 2.1 mm i.d.) with a Sentry guard column (3.5 μm, 10 × 2.1 mm i.d.) both supplied by Waters (Zellik, Belgium). The column was kept at room temperature. A mobile phase consisting of eluents A [water/methanol/acetic acid (94/5/1, *v*/*v*/*v*) containing 5 mM ammonium acetate] and B [methanol/water/ acetic acid (97/2/1, *v*/*v*/*v*) containing 5 mM ammonium acetate] was used at a flow rate of 0.3 mL min−1. A gradient elution was applied as follows: 0–7 min, 95% A/5% B –35% A/65% B; 7–11 min, 35% A/65% B–25% A/75% B; 11–13 min, 25% A/75% B–0% A/ 100% B; 13–15 min, 0% A/100% B; 15–16 min, 0% A/ 100% B–40% A/60% B; 16–22min, 40% A/60% B–60% A/40% B; 22–23 min, 60% A/40% B–95% A/5% B; 23–25 min 95% A/5% B. The injection volume was 20 μL.

#### *5.5. Calculation of Mycotoxin Adsorption Rate (%)*

The percentage of adsorption rate was calculated according to the following equation:

$$\% \text{ binding capacity} = 100 \times (\text{Ci} - \text{Cf}) \text{Ci} \tag{1}$$

where

Ci is the initial concentration of mycotoxin

Cf is the concentration of unbound mycotoxin after incubation period

#### *5.6. Calculation of the Binding E*ffi*ciency*

The binding efficiency of the clays was determined according to the obtained results of the adsorption and desorption rate ((1) and (2))

% Desorption = ((% Adsorption pH3 − % Adsorption pH7)/% Adsorption pH3) × 100 (2)

with

$$\% \text{ binding efficiency} = \% \text{ adsorption} - \% \text{Designtion} \tag{3}$$

#### *5.7. Statistical Analysis*

Data were analyzed using the statistical software SPSS version 24 (IBM, New York, NY, USA) and results are presented as means ± SD. All analyses were performed in triplicate, and the significance of the means value was performed with the student's t-test after determination of normality. Results were considered statistically significant at *p* ≤ 0.05.

**Author Contributions:** R.R. perform the extraction, preparation, and characterization of clays, contribute in the mycotoxin binding experiment and was involved in all aspect of this study. G.A. secured, premise and set the mycotoxin binding experiment and reviewed the paper. M.D.B. and S.D.S. reviewed data of the mycotoxin binding experiment and reviewed the paper. M.V.d.V. performed the experiment of the mycotoxin binding capacity. C.D. collected the data of the mycotoxin binding experiment. R.D. supervised editing and reviewed the entire work. M.H.A. who proposed the initial idea of studying the characteristics of the Tunisian clays and their efficiency in poultry feeds, helped in performing the characterization of clays and reviewed the paper. A.G. supervised the preparation and a part of the characterization of clays.

**Funding:** This research received no external funding.

**Acknowledgments:** We would like to express our sincere appreciation and gratitude to Professor Maher Radaoui and Sana Ncib who helped us in doing the BET and DRX analyses of clays. We would like to thank Imen Yehmed for doing the XRF analyses of clays. The authors acknowledge the support of MYTOX-SOUTH (https://mytoxsouth.org).

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

#### **References**

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

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**9. Expanding transcriptome data to aid development of molecular markers**

A DNA-based molecular marker is a DNA sequence with an identifiable location on the genome that can be transmitted from one generation to the next following the standard laws of inheritance [87]. Recent years have witnessed an immense interest in generation and utilization of molecular markers, as they provide the essential tools for a variety of genomic applications such as QTL mapping, map-based cloning, marker-assisted breeding, association mapping and genetic diversity assessment. These approaches can be applied to understand the genomic architecture of the crop and can expand the efficiency of breeding programmes, thereby aiding to expedite agricultural research. The advent of NGS has enabled the exploration of thousands of markers across entire genomes and transcriptomes. Although transcriptomics has been majorly used for gene expression analysis, it has also been utilized to identify molecular markers such as SSRs and SNPs especially in the genic regions. Such gene-based markers located in coding regions of the genes greatly enhance the opportunity of precise mapping of genes linked to important traits. Transcriptome sequencing offers another advantage for those crops in which a reference genome is not available. To identify SSRs from transcriptome data, several bioinformatics tools have been developed such as MISA (MIcroSAtellite identification tool) (pgrc.ipk-gatersleben.de/misa/), RISA (Rapid Identification of SSRs and Analysis of primers) (http://sol.kribb.re.kr/RISA/) and RepeatAnalyzer [88]. In chickpea, initially a large number of molecular markers were derived from ESTs. Buhariwalla et al. [89] reported 106 EST-based markers developed from an EST library of root tissue from chickpea. In another study by Choudhary et al. [90], 2131 ESTs were utilized for development of 246 EST-SSR markers. Apart from SSRs, several types of markers such as ESTPs, PIPs and EST-SNPs were developed in chickpea using transcriptome data. For instance, Choudhary et al. [91] reported 125 EST-SSRs, 109 ESTPs, 102 SNPs and 151 ITPs. Gupta et al. [57] reported 367 novel EST-derived functional markers which included 187 EST-SSRs, 130 potential intron polymorphisms (PIPs) and 50 expressed sequence tag polymorphisms (ESTPs). In another study, 71 gene-based SNP markers were developed utilizing candidate chickpea transcripts [92]. However, currently transcriptomic resources can be easily generated by high-throughput NGS technologies and utilized to identify molecular markers very rapidly and cost-effectively. Hiremath et al. [36] utilizing the Roche platform generated about 3000 gene-based markers from a large subset of transcripts derived from different chickpea tissues. Currently, SNPs are the markers of choice and are preferred over the SSRs and other markers because of their genome-wide presence and amenability to high-throughput genotyping. Theoretically, SNP calling may be defined as the process of identifying a single-nucleotide variation from an accession read that differs from the existing reference genome or a de novo assembly at similar nucleotide position. Read assembly files generated by mapping programs such as BWA, Bowtie and SOAP are used to perform SNP calling. Bioinformatics tools such as HaploSNPer [93], SAMtools [94, 95], POLYBAYES [96], SNVer [97] and SOAPsnp [98] have been designed to detect the variations in the NGS data. Comparison of transcriptome datasets from contrasting genotypes could help derive SNPs. To date, several studies have been carried out using NGS technology-based transcriptome sequencing to generate large sets of molecular markers in various crop species including chickpea. For instance, a report by Garg et al. [35] facilitated identification of 4816 SSRs from the de novo assembly of the chickpea transcriptome. In another study, sequencing the transcriptome of *C. reticulatum* (PI489777), the wild relative of chickpea, by GS-FLX 454 technology, generated a total of 4072 SSRs and 36,446 SNPs.

Likewise, Agarwal et al. (2012) sequenced the transcriptome of ICCV2 and identified 5409 SSRs. Amongst these, 130 and 493 SSRs were found to be polymorphic after comparing with the transcriptome of *desi* and *wild* chickpea. In addition to the SSRs, a total of 1986 and 37,954 SNPs were also identified between the desi, kabuli and wild genotypes. Similarly, in another study, 51,632 genic SNPs were identified by 454 transcriptome sequencing of *C. arietinum* and *C. reticulatum* genotypes [99]. In a recent study, Srivastava et al. [100] identified 11,621 differentially expressed genes in root vs shoot tissues using RNA sequencing of a wild perennial *Cicer microphyllum* and integrated above transcriptome profiling with high-resolution QTL mapping in order to identify drought-responsive root-specific genes. The transcriptomic resources, therefore, clearly have remarkable potential to expedite the development of large numbers of molecular markers which can be used in genomic-assisted breeding for develop-

Transcriptome Analysis in Chickpea (*Cicer arietinum* L.): Applications in Study of Gene...

http://dx.doi.org/10.5772/intechopen.69884

255

The last few years have witnessed legume genomics attaining new heights as genomes, and transcriptomes of many model legumes (*M. truncatula*, *L. japonicus*) and crop legumes (*G. max*, *C. cajan*, *P. vulgaris*, *Arachis hypogaea*, *Vignas*, etc.) were sequenced. Transcriptomes of both types of cultivated chickpea (desi and kabuli) and its wild progenitor (*C. reticulatum*) have also been sequenced. Several studies have been carried out to analyse the transcriptome of chickpea which have led to genome-wide determination of transcript levels in various tissues and developmental pathways as well as during biotic and abiotic stresses. This comprehensive analysis of the chickpea transcriptome has advanced the understanding of the molecular mechanisms underlying several critical biological pathways in chickpea. Moreover, analyses of the non-coding RNAs have revealed potential regulators of important pathways affecting the overall development and stress tolerance in chickpea. Further, transcriptome analysis has also facilitated the development of large sets of genic molecular markers such as SSRs and SNPs that will serve as excellent tools for advancing the chickpea breeding programmes. Overall, the transcriptome sequencing of chickpea has not only provided a deep insight into the gene space and quantitation of gene expression but also an opportunity to isolate genes of

Chandra Kant, Vimal Pandey, Subodh Verma, Manish Tiwari, Santosh Kumar and Sabhyata

interest and functional markers for use in chickpea improvement.

\*Address all correspondence to<EMAIL_ADDRESS>
National Institute of Plant Genome Research, New Delhi, India

ing improved varieties of chickpea.

**10. Future perspectives**

**Author details**

Bhatia\*

Likewise, Agarwal et al. (2012) sequenced the transcriptome of ICCV2 and identified 5409 SSRs. Amongst these, 130 and 493 SSRs were found to be polymorphic after comparing with the transcriptome of *desi* and *wild* chickpea. In addition to the SSRs, a total of 1986 and 37,954 SNPs were also identified between the desi, kabuli and wild genotypes. Similarly, in another study, 51,632 genic SNPs were identified by 454 transcriptome sequencing of *C. arietinum* and *C. reticulatum* genotypes [99]. In a recent study, Srivastava et al. [100] identified 11,621 differentially expressed genes in root vs shoot tissues using RNA sequencing of a wild perennial *Cicer microphyllum* and integrated above transcriptome profiling with high-resolution QTL mapping in order to identify drought-responsive root-specific genes. The transcriptomic resources, therefore, clearly have remarkable potential to expedite the development of large numbers of molecular markers which can be used in genomic-assisted breeding for developing improved varieties of chickpea.