**Entropy in Foundations of Quantum Physics**

Special Issue Editor **Marcin Pawłowski**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Special Issue Editor* Marcin Pawłowski University of Gdansk ´ Poland

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Entropy* (ISSN 1099-4300) (available at: https://www.mdpi.com/journal/entropy/special issues/ Foundations Quantum Physics).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Article Number*, Page Range.

**ISBN 978-3-03928-951-6 (Pbk) ISBN 978-3-03928-952-3 (PDF)**

c 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND.


*Article* **Optimization of Fuzzy Controller for Predictive Current Control of Induction Machine**

**Toni Varga** *<sup>∗</sup>***,†, Tin Benši´c †, Marinko Barukˇci´c and Vedrana Jerkovi´c Štil**

Faculty of Electrical Engineering, Computer Science and Information Technology Osijek, Kneza Trpimira 2b, 31000 Osijek, Croatia<EMAIL_ADDRESS>(T.B<EMAIL_ADDRESS>(M.B<EMAIL_ADDRESS>(V.J.Š<EMAIL_ADDRESS>
 †Theseauthorscontributedequallytothiswork.

**Abstract:** An optimization procedure for type 1 Takagi–Sugeno Fuzzy Logic Controller (FLC) parameter tuning is shown in this paper. Ant colony optimization is used to obtain the optimal controller parameters, and only a small amount of post-optimization parameter adjustment is needed. The choice of controller parameters is explained, along with the methodology behind the criterion for objective function value calculation. The optimized controller is implemented as an outer-loop speed controller for Predictive Current Control (PCC) of an induction machine. The performance of the proposed control method is compared with that of several other model predictive control methods. The results show a 55% decrease in speed tracking error and 74% decrease in torque overshoot.

**Keywords:** induction machine; ant colony optimization; predictive current control; fuzzy logic control; Takagi–Sugeno


*3.1. PhotoStress Method*

The PhotoStress method is a non-contact full-field measurement technique used to determine surface strains and transform them into stresses occurring in a structure. To use this method, a special strain-sensitive plastic coating needs to be bonded to the analyzed structure. Subsequently, after applying the loading to the structure, its strains are transmitted to the coating assuming the same strain condition as the part of the structure, which it is bonded to. After illuminating the coating by polarized light emitted from a reflection polariscope, a colorful pattern that is viewed through the polariscope occurs due to strain. There are two types of color pattern investigated known as isoclinic and isochromatic fringes. At every point on an isoclinic, the directions of principal strains are parallel to the direction of the analyzer and polarizer polarization. The photoelastic strain pattern appears as a series of successive and contiguous, variously colored bands known as isochromatic fringes representing a different level of the principal stresses difference [29,30]. When the loading is applied to the structure, the coated part color changes from black characterizing the no-loading state of the structure and first colors appear in the areas of the highest stress. After increasing the loading, the color fringes spread throughout the coated part (Figure 10), additional fringes are generated in the highly stressed areas of the investigated structure and move towards the regions of zero or low stress until the maximum load is achieved.

**Figure 10.** Characteristic change of the isochromatic fringes observed in PhotoStress method during increasing uniaxial loading.

The quantitative analysis of principal strain difference (strain intensity) occurring at any point of the coating can be quickly performed using a digital compensator attached to the polariscope. Strain intensity analysis depends only on the recognition of the fringe order defined by a color and understanding of the relationship between the fringe order and strain intensity as follows:

$$
\varepsilon\_1 - \varepsilon\_2 = N \cdot \frac{\lambda}{2 \cdot t\_c \cdot K} = N \cdot f \tag{1}
$$

where *ε*1,*ε*2 are the principal strains, *N* is the fringe order, *λ* is the wavelength of the light emitted by polariscope, *tc* is the thickness of the coating, *K* is the strain optical coefficient of the coating and *f* is the fringe value of coating.

The advantage of the PhotoStress method is its high sensitivity for small strain/stress levels. On the other hand, in the areas with high strain/stress gradient, a problem can occur by quantifying the values (for the higher-order fringe recognition, the microscope is required to be used).

### *3.2. Digital Image Correlation Method*

The deformation analysis was also carried out by a low-speed digital image correlation system Q-400 (Dantec Dynamics A/S, Skovlunde, Denmark) working on the principle of the DIC method (some detailed information are presented in Appendix B). The digital image correlation principle is based on the correlation of digital images captured during loading of the analyzed object, where the images are not compared as whole units, but as small image elements called facets. The shape of the facet used by correlation systems Dantec Dynamics is squared, and each facet commonly comprises a group of pixels ranging in size from 15 × 15 px to 30 × 30 px. Depending on the type of analysis, the facet size can be adjusted (reduced or enlarged). The facets may touch or overlap, but there must not be an empty area between them. Since the information about the displacements of the analyzed object is obtained at the nodes of the virtual grid, which correspond in position to the centers of the facets, the overlap of the facets (Figure 11) is one way to increase the data resolution, i.e., to obtain a more considerable amount of data and, thus, to better reconstruct the surface of the analyzed object (especially around the edges). The manufacturer of correlation devices, Dantec Dynamics, recommends overlapping the facets up to 1/3 of the facet size because with such an overlap the data points are still independent.

**Figure 11.** Illustrative example of the increase of the data resolution caused by the facet overlap: (**a**) defined area of interest; (**b**) measuring points (green dots) when the facets are in touch, (**c**) measuring points (green dots) when the facets are overlapped.

Dantec Dynamics correlation devices use an algorithm based on a pseudo-affine transformation to obtain information on the transformation coordinates of the analyzed object surface points. If transformation parameters of possible displacement, elongation, shear, and distortion of the facet *a*0 − *a*7 as shown in Figure 12 are considered, using the aforementioned algorithm the transformation coordinates (*u*, *v*) can be calculated as follows:

$$\begin{array}{l} u(a\_0, a\_1, a\_2, a\_3, \widetilde{\mathbf{x}}, \widetilde{\mathbf{y}}) = a\_0 + a\_1 \cdot \widetilde{\mathbf{x}} + a\_2 \cdot \widetilde{\mathbf{y}} + a\_3 \cdot \widetilde{\mathbf{x}} \cdot \widetilde{\mathbf{y}},\\ v(a\_4, a\_5, a\_6, a\_7, \widetilde{\mathbf{x}}, \widetilde{\mathbf{y}}) = a\_4 + a\_5 \cdot \widetilde{\mathbf{x}} + a\_6 \cdot \widetilde{\mathbf{y}} + a\_7 \cdot \widetilde{\mathbf{x}} \cdot \widetilde{\mathbf{y}},\end{array} \tag{2}$$

where (*x*, *y*) *T* are the lens-distorted 2D coordinates of the point in the normalized image plane [31].

**Figure 12.** Transformation parameters used in the algorithm based on pseudo-affine transformation.

There are some relevant differences between the measurements of relieved deformations performed by 2D DIC and 3D DIC, respectively. Firstly, if the hole-drilling causes normal displacement of the analyzed specimen surface (this displacement component cannot be analyzed by a single-camera system), it leads to a correlation error and affects

the analysis results. Another advantage of the 3D correlation system is the possibility to measure all the three displacement components not only of a flat but also curved specimen. Higher distortion of the lenses, which are not directed perpendicularly to the analyzed object surface, can be considered a small drawback of the 3D systems. Also, the requirement for a proper arrangement of the cameras towards the analyzed object means that the analysis performed by the 3D correlation system is done from a greater distance to the specimen. However, this limitation can be reduced using quality CCD cameras with sufficient image resolution of the sensors.

The accuracy of the results of the deformation analysis for both types of the analysis (2D or 3D) can be affected, for example, by:

• The quality of the speckle-pattern created on the analyzed object surface and illumination

For correct image correlation, it is essential to make sure that each facet is unique, i.e., it contains a unique pixel distribution with different levels of intensity of grey color. As the aim of the analysis was to use image resolution of the sensors as well as possible and to perform evaluation of the results near the edge of the drilled hole, it was necessary to create the black-and-white speckle-pattern with very fine speckles (Figure 13). The illumination of the specimen surface should be homogenous and reach high sharpness and sufficient contrast of speckles. Compliance with this requirement was ensured using the Dedolight DLH400DT (Dedo Weigert Film GmbH, München, Germany) halogen reflector with white light.

**Figure 13.** Pairs of digital images captured by 3D Q-400 Dantec Dynamics: (**a**) reference image; (**b**) evaluated image with a defined mask of evaluation.

• Calibration parameters

The correctness of the coordinates transformation of the object points from 3D world coordinate system into 2D sensor system depends on the accuracy of the obtained external (i.e., mutual position and rotation of the cameras) as well as internal (focal length, principal point coordinates, tangential and radial distortion of the lenses) calibration parameters of the cameras. The calibration of Dantec Dynamics correlation systems is automatized, and based on the Zhang algorithm [32]. Information about the calibration accuracy is provided by the so-called calibration residuum, whose value should not exceed 0.5 px. The size of the calibration target should approximately correspond to the size of the analyzed specimen. Therefore, calibration of the stereo-camera correlation system was done using a calibration target comprising 9 × 9 checkboard fields with a precisely defined distance of 3 mm. The residuum obtained by the cameras calibration reached the value of 0.279 px, which is, according to the aforementioned criterion, considered as accurate calibration.

• Correlation parameters and the related levels of smoothing

Both Q-400 Dantec Dynamics cameras captured the digital images with image resolution of 1800 × 2056 px. The pixel density was approximately 64 px/mm. During the

analysis a couple of reference images (Figure 13a) captured by the maximum loading force of 250 N were taken in the Istra4D ver. 4.3.0 control software. The pair of images capturing the deformation of the analyzed specimen surface area after drilling of the hole (Figure 13b), was correlated with the reference one according to the following aspects. The facet size as one of the correlation parameters set up by the evaluation of the measurement was adapted to the fact that the drilled hole's edge would be reconstructed as accurately as possible if the facet was as small as possible. However, facets are required to be unique, i.e., all the facets have to comprise randomly distributed pixels with a high range of gray values. Therefore, the measurement was evaluated with the facet size set to 23 × 23 px, and their overlapping of 6 px ensured the increase of the data points (points in which the displacements and strains were evaluated) resolution.

The results of the deformation analysis performed by the Dantec Dynamics correlation systems are due largely to the properly set smoothing level. Istra4D ver. 4.3.0 contains two types of smoothing. The first known as *local regression* should be used mainly in cases if a high deformation gradient is expected in the deformation analysis results. The second is called *smoothing spline* and is used to smooth the deformation field with approximately homogeneously distributed deformation levels. As with the hole-drilling method the stress concentrator occurs in the specimen, the authors used a filter of local regression. Several studies described, e.g., in [33,34] were conducted on the proper setting of the local regression level. For the illustration, the authors point to the effect of local regression in Figure 14, which shows the relieved strain intensity fields obtained by different levels of smoothing. Figure 14a shows the relieved strain intensity field obtained by default settings of smoothing, i.e., without smoothing. The other three relieved strain intensity fields correspond to the results obtained by the settings of kernel size to 7 × 7 (Figure 14b), 15 × 15 (Figure 14c), and 31 × 31 (Figure 14d).

**Figure 14.** Influence of smoothing on the strain intensity field obtained by Q-400: (**a**) without loading; (**b**) kernel size of 7 × 7; (**c**) kernel size of 15 × 15; (**d**) kernel size of 31 × 31.

According to the manufacturers of Dantec Dynamics correlation systems, the strains are computed only from the local curvatures of facets when the kernel size is set to 3 × 3. The higher the kernel size, the higher the influence of the deformation gradient on the strains. For the kernel size set to 9 × 9 up to 31 × 31 (i.e., the highest level of smoothing based on local regression in Istra4D ver. 4.3.0), the strains are computed only from the deformation gradients. According to the authors' analysis, the kernel size of 15 × 15 for which the results obtained are presented in Figure 14c corresponds to the optimal setting of smoothing for the described type of evaluation. The effect of oversize smoothing can be observed in Figure 14d.

#### **4. Results**

Although the aforementioned measurement was conducted by two different noncontact optical methods providing the full-field information about the strains, the quantitative comparison of the results obtained from both techniques is not simple. While the results of the measurement by the DIC method provide quantitative information about

the displacements as well as strains in the center of each facet, for the quantification of the results obtained using PhotoStress method, the use of a digital compensator is necessarily required in each evaluated location of the specimen.

In many cases, experimental testing results serve to verify the results obtained numerically, e.g., using software based on the finite element method (FEM) [35]. The abovedescribed strain analysis in the vicinity of the hole drilled in the flat specimen loaded by uniaxial tension, is a typical analysis performed by the finite element analysis (FEA). For that reason, the authors reversed the standard approach and, thus, verified the experimentally obtained results by numerical analysis. Such an approach has already been used by the authors, and the results obtained were published in several scientific publications, e.g., [36–39]. Moreover, in conventional quantification of residual stresses by the hole-drilling technique the FEA is usually used, e.g., the correlation parameters *a*, *b* used in the formulas for the computation of stress components from the relieved strains are determined mainly in a numerical way.

#### *4.1. Finite Element Analysis*

The numerical model of the analyzed specimen was created in Abaqus/CAE 2020 (SIMULIA, Johnston, RI, USA) software. The analysis was focused on the determination of the relieved displacements/strains occurring in the specimen (with dimensions, mechanical properties, constraints and loading described in head 3) after milling a through-hole lying on its longitudinal axis. Detailed information on the procedure of numerical analysis is presented in Appendix C.


*2.1.1 Fluorescent coumarin succinimidyl esters*

Succinimidyl esters (SE) are proven to be very good reagents for amine modifications. These kinds of reagents are generally stable and show good reactivity and selectivity with aliphatic amines, such as the amine group of lysine side chain. Some of these kinds of reactive dyes are hydrophobic molecules and should be previously dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO), but the sulfo-succinimidyl esters are water soluble. The amine labelling

**Figure 1.**

*Schematic diagram of amine labelling techniques using succinimidyl esters (A), 4-sulfotetrafluorophenyl esters (B), sulfonyl chlorides (C), and isothiocyanates (D).*

reaction with succinimidyl esters has a handicap, due to its great pH dependence. Succinimidyl esters react with non-protonated aliphatic amine groups, and the amine acylation reaction must be carried out at pH > 7.5. In the specific case of protein labelling by succinimidyl esters, the reactions require a pH between 7.5 and 8.5. Buffers used in labelling reactions shall not contain nucleophilic compounds because they may react with the labelling reagent to form unstable intermediates that could destroy the reactive dye. Most conjugations are done at room temperature, but either high or low temperature may be required for a particular labelling reaction. Some of the fluorescent coumarin succinimidyl esters contain a seven-atom aminohexanoyl spacer between the fluorophore and the reactive group, providing better solubility and spatial separation between the fluorophore and the target molecule being labeled. This separation potentially reduces the quenching that typically occurs upon conjugation and makes the dye more available for recognition by secondary detection reagents [68]. The most important fluorescent coumarin succinimidyl esters used for labelling biomolecules are shown in **Table 1**, as the corresponding values of maximal excitation (Ex) and emission (Em) wavelengths and their physicochemical features and biological applications [19, 68].


**1. Introduction**

Polyoxometalates (POMs), a class of metal oxide clustered anions, have already been investigated for more than 200 years. Their history dates back to 1826, when Berzelius reported the discovery of the first POM cluster (NH4)3[PMo12 O40] nH2O [1]. However, due to di fficulties achieving insights into the POM structure, no significant progress was made until Keggin determined the structure of H3PW12 O40 in 1934 [2]. Since then, the interest of scientists in POMs has increased drastically, not only in the development of new POM structures, but also towards their use in various applications, such as catalysis, optics, magnetism, biological medicine, environmental science, life science, and technology [3–7]. In particular, their use in catalysis is one of the most examined fields because of their strong acidity, oxygen-rich surface, photoactivity, and redox capability. Despite these interesting characteristics, POMs still exhibit some drawbacks for their use in catalysis. First, POMs possess a low surface area (<10 m2 g−1), which consequently hinders the accessibility of reactants and secondly, their high solubility in aqueous solutions and polar organic solvents results in a low recyclability [4]. The immobilization of POMs into/onto porous solids has been proposed to overcome these shortcomings and to achieve catalysts with a high catalytic performance. In the past few decades, many porous materials have been examined as supports for the immobilization of POMs, e.g., silica, ion-exchange resin, zeolites, and activated carbon [5–8]. Since the discovery of metal–organic frameworks (MOFs), much e ffort has been dedicated to use these porous materials as potential supports for POMs. MOFs are inorganic–organic hybrid crystalline materials that are constructed from metal ions or clusters and organic linkers through coordination bonds. These materials have attracted considerable interest in recent years due to their large surface areas, tunable pore size, and designable functionalities. So far, MOFs have shown great potential in gas storage and separation, catalysis, sensing, drug delivery, proton conductivity, solar cells, supercapacitors, and biomedicine [8–13]. Moreover, MOFs are regarded as an outstanding platform for introducing guest molecules because of the high accessibility of their internal surface area and long-range ordered channels. So far, several active sites have been successfully embedded in the pores or cages of MOFs, such as noble metals, metal oxides, enzymes, and POMs [14–17].

The first report of a POM@MOF hybrid was reported in 2005 by Férey and co-workers [18]. In this seminal work, the POM, K7PW11O39 (van der Waals radius, 13.1 Å), was successfully encapsulated into the big cages of the highly stable Cr-based MOF, MIL-101, by using an impregnation method. To date, several other thermal and chemical stable MOFs have been applied as supports to host POMs for their use in catalysis, including MIL, UiO, ZIF series, NU-1000, and Cu-BTC frameworks (see Table 1). The most examined POMs that have been encapsulated into MOFs are the well-known Keggin [XM12O40]n− and Dawson [X2M18O62]n− (X = Si, P, V, Bi, etc.; M = V, Mo, W, etc.) POMs and their derivatives. These POMs are of significant interest because their structure and properties can be easily varied by removing one or more MO4+ units, leading to lacunary POMs such as [PW9O34]9−, or by the substitution of X and M by different metals or a combination of two fragments of the Keggin structure, leading to sandwich-type POMs such as [Tb(PW11O39)2]11−.

There are several advantages of using MOFs as a host matrix to encapsulate POMs. First of all, their exceptionally high surface areas and confined cages/channels make it possible to ensure a homogeneous distribution of the POM in the MOF host. This not only prevents the agglomeration of POMs, but also improves their stability and recyclability and ensures a fast diffusion of substrates and products. Secondly, the highly regular cages and windows of MOFs ensure a high substrate selectivity, or, in other words, only specific substrates/products are able to reach the active POM sites. Thirdly, owing to the good interaction and electron transfer between the MOF and POM, an increased synergistic catalytic performance is typically observed. Finally, the chemical environment of POMs can easily be adjusted through modification or functionalization of MOFs. Therefore, POM@MOF hybrids not only combine the interesting properties of POMs and MOFs, but also allow the aforementioned disadvantages of POMs to be tackled to afford synergistic catalysis. This review is focused on the synthetic aspects of POM@MOF hybrids, as well as their use in catalysis (organocatalysis, electrocatalysis, and photocatalysis). Alongside the POM@MOF systems discussed here, where the POMs are encapsulated inside a MOF host, POM-based MOFs have also been investigated. In these MOFs, the POMs form the actual metal nodes that are interconnected by organic linkers [19–21]. However, these fall outside the scope of this review.

**Table 1.** Physical properties of representative metal–organic frameworks (MOFs) used to encapsulate polyoxometalates (POMs) for catalysis.

#### **2. Synthesis and Design of POM@MOF**

To date, several well-known highly stable MOFs have been used to encapsulate POMs, including MIL, UiO, and ZIF series, as well as NU-1000 and Cu-BTC frameworks. One of the most commonly applied methods to embed POMs in MOFs is impregnation. Wet impregnation is a simple and straightforward method, since most of the POMs are well-soluble in polar solvents. Typically, the activated MOF powder is immersed in the POM solution to obtain the composite material. Several POM@MOF hybrids have been successfully synthesized through this wet impregnation method, such as POM@MIL, POM@ZIF, and POM@UN-1000. An important aspect allowing the use of this method is that the size of the POM must be smaller than the windows of the MOF. Moreover, for some POMs and MOFs, it was observed that the POM loading could not be enhanced by increasing the concentration of POM in aqueous solution when a certain POM loading was achieved. For example, for POMs whose size is bigger than the pentagonal windows (12 Å) of MIL-101(Cr), the POMs were only encapsulated into the large cages, while the other cages, which represent 2/3 of the total number of cages of MIL-101(Cr), were unfilled. Naseri et al. demonstrated that the loading of a sandwich-type POM [(HOSnOH)3(PW9O34)2]12− (15.2 Å × 10.4 Å) could not be enhanced by increasing the concentration of POM in the aqueous solution [29].

The impregnation method cannot be used for MOFs whose window size is smaller than the POMs, e.g., Cu-BTC, UiO, and ZIF. Therefore, for these MOFs, the one-pot (also known as bottle-around-the-ship) synthesis method has been applied to obtain POM@MOF hybrids. The one-pot method is also often used to obtain POM-encapsulated MOFs in which the anionic form of the POM acts as a structure directing agent to ensure deprotonation of the organic carboxylate ligand. For the preparation of POM@MOF hybrids, typically, the synthesis parameters employed to obtain the parent MOF are used upon addition of the POM. The one-pot method can not only be used to synthesize POM@MOFs that cannot be obtained by impregnation, but can also confine the POMs in the MOF cages to prevent leaching if the size of the POMs is bigger than the windows of the MOFs.

Therefore, in conclusion, the synthesis approaches commonly used to incorporate POMs into MOFs are impregnation and one-pot synthesis. To choose, however, the "correct" methodology, two questions need to be addressed in advance: does the size of the POM fit into the MOF cages and can the pore window of the MOF confine the POM? If both criteria are met, one can expect that the obtained catalyst will work e fficiently at a molecular level.

As was mentioned before, the first report on the embedding of a POM into the cages of an MOF was reported by Férey's group. They showed that a Keggin-type POM, K7PW11 O40 (van der Waals radius, 13.1 Å), can be confined in MIL-101(Cr) by simple impregnation. The resulting MIL-101-Keggin solid was characterized by XRD, TGA, and N2 sorption, as well as IR and 31P solid state NMR, which confirmed the presence of Keggin ions within the pores [18]. As summarized in Table 1, MIL-101 has two types of mesoporous cages: a smaller one with an inner size of ~29 Å and pentagonal windows of ~12 Å, and a larger one with an inner size of ~34 Å and hexagonal windows of ~15 Å. Based on the size of the cage windows and the size of the POM, one can conclude that the POM can only diffuse into the largest cages.

In 2010, Gascon and co-workers prepared PW12@MIL-101 (PW12 = [PW12 O40] 3−) composites by using a one-pot and wet impregnation method [30]. The authors observed a homogeneous distribution of PW12 when the one-pot synthesis was applied under stirring conditions. By using the wet impregnation method, high loadings of PW12 in MIL-101 resulted in a drastic decrease in the surface area and pore volume. However, this decrease in surface area and pore volume was smaller for the one-pot synthesis method in comparison to the impregnation method using the same PW12 loading. The authors stated that in the one-pot synthesis, both the large- and medium-sized cavities were occupied, while, when using the impregnation method, only the larger cavities were accessible.

Canioni and co-workers compared di fferent synthesis methods for encapsulating POMs in MIL-100(Fe) [31]. The authors observed a good agreement between the experimentally obtained POM loading and the maximum theoretical loading for the PMo12@MIL-100 (PMo12 = [PMo12 O40] 3−) obtained by a one-pot solvothermal synthesis. In addition to this, the solvothermally obtained

PMo12@MIL-100 showed a good stability in aqueous solution and no POM leaching was observed after 2 months. On the contrary, the PMo12@MIL-100 material prepared through impregnation exhibited significant POM leaching after 2 months.

Based on the above examples, POM leaching was observed for the POM@MIL-101 and POM@MIL-100 obtained by impregnation, since immobilization is based on an adsorption equilibrium. One way to circumvent this leaching is to use amino-functionalized MOF structures, e.g., UiO-66-NH2 and MIL-53-NH2, which can ensure a better interaction with the polyanions [32–34]. The formation of complexes such as −NH3 +[H2PW12 O40] − between primary amines, ammonia, or pyridine and PW12 is well-documented [35]. In 2012, Gascon and co-workers used a microwave-assisted one-pot synthesis to obtain PW12@MIL-101-NH2(Al) as their attempts to synthesize MIL-101-NH2(Al) containing PW12 by one-pot solvothermal synthesis were not successful [36]. One year later, Bromberg et al. examined the encapsulation of POMs in amino-functionalized MOFs (NH2-MIL-101(Al) and NH2-MIL-53(Al)) by immobilization. They concluded that POMs electrostatically interact with the MOF surface to form a stable composite. The thermal stability of the composites PW12@NH2-MIL-53(Al) and PW12@NH2-MIL-101(Al) was similar to the stability of the parent MOFs [37].

Besides MIL-101, Cu-BTC (namely HKUST-1 or MOF-199) has also been used as a host material to encapsulate Keggin- and Dawson-type POMs [38,39]. As shown in Table 1, the larger cages of Cu-BTC have an inner diameter of 1.3 nm and a pore window of 0.9 nm, which perfectly ensures the stable entrapment of POMs. For example, PW12 with a diameter of approximately 1.06 nm was used as a structure directing agent for the self-assembly of Cu-BTC at room temperature [38]. The authors observed an enhanced chemical and thermal stability after the embedding of POM and no POM leaching was noted during catalysis in several studies [40,41]. In one of these studies, Shuxia Liu's group prepared a series of Keggin-type POMs in Cu-BTC, denoted as NENU-n, n = 1~10, and formulated as [Cu2(BTC)4/3(H2O)2]6 [H*n*XM12 O40]·(C4H12N)2 (X = Si, Ge, P, As, V, Ti; M = W, Mo), by using a one-pot hydrothermal synthesis [42,43]. The templating effect of the POMs resulted in highly crystalline composite materials which showed an enhanced thermal stability. Moreover, as large crystals were obtained, the structures were elucidated by means of single-crystal X-ray diffraction, demonstrating that the Keggin polyanions were confined in the larger cuboctahedral cages (inner diameter of 1.3 nm) [43].

Besides MIL-101 and Cu-BTC, isostructural imidazolate frameworks, namely ZIF-8 and ZIF-67, have also been frequently used as the host matrix. The sodalite-type cavities of ZIF-8 have a size of approximately 1.1 nm, but the accessible window of the cavity is rather small (0.34 nm). Keggin-based POMs possess a relatively larger particle size up to 1.3 ~ 1.4 nm in comparison to the cavities of ZIF-8, but can fit perfectly in their anionic form (1 nm diameter of PW12) [33]. Therefore, the bottle-around-the-ship method is an ideal approach for confining POMs inside ZIF-8 or ZIF-67 [44]. For instance, Malka et al. reported a POM encapsulated in ZIF-8 for its use as an esterification catalyst. The authors were able to obtain a PW12 loading of 18 wt% by using a one-pot synthesis strategy at room temperature in aqueous solution. However, after three catalytic cycles, degradation of the MOF occurred and 9% of the POM leached out [45]. A way to overcome the POM leaching in ZIF-8 was demonstrated in the work of Jeon et al. In this study, an impregnation method was used to functionalize the surface of the ZIF-8 nanoparticles with a Keggin-type PW12, in order to obtain a core–shell MOF–POM composite. Interestingly, due to the strong interaction, the POM-decorated MOF became insoluble in hydrophilic solvents [46].

Lin and co-workers constructed a POM@MOF molecular catalytic system with a Ni-containing POM [Ni4(H2O)2(PW9O34)2]10− (namely Ni4P2) into an [Ir(ppy)2(bpy)]+-derived MOF by one-pot synthesis, and the MOF was isostructural to UiO-66, with extended ligands. Ni4P2 POMs can be encapsulated in the octahedral cages with an inner dimension of 2.2 nm [47].

In the studies mentioned above, the one-pot synthesis and wet impregnation methodology both give a high chance of success in the synthesis of POM-encapsulated MOFs. Although the impregnation method is straightforward, it is only applicable for MOF pore windows larger than the POMs. However, leaching of the POMs might happen unless precautions are taken in advance to ensure a good interaction between POM and MOF supports. The use of POMs as a template might enhance the crystallinity of the MOF framework, which makes a one-pot synthesis very attractive. However, it is important to note here that the size of the POM needs to be larger than the pore window of the MOFs to prevent leaching. In addition to this, the one-pot synthesis method is not applicable to all MOF structures. In most cases, the POM@MOFs materials obtained through a one-pot synthesis or impregnation method have a positive influence on the thermal stability in comparison to the parent non-functionalized MOF.

Besides the commonly used one-pot and impregnation method, there are some other efficient methods for constructing POM-encapsulated MOFs. In 2018, Zhong et al. synthesized NENU-3 (PW12@HKUST-1) by a liquid-assisted grinding method [48]. By using a two-step synthesis, PW12 and the Cu salt were first dissolved and evaporated to obtain the copper salt of PW12. Hereafter, the H3BTC ligand was added in the presence of a small amount of alcohol (MeOH and EtOH), which was used as the grinding liquid. The mixture was ground for 5 min and the color gradually changed to blue. After washing and drying at 60 ◦C for 24 h, the obtained nanocrystalline, NENU-3, showed a high crystallinity, and the surface area was slightly higher compared to NENU-3 obtained in one-pot solvothermal synthesis. In 2019, G. Li et al. employed an in-situ hot-pressing approach to encapsulate the Keggin-type PW12 into an indium-based MOF (MFM-300(In)) [49]. As shown in Figure 1, all the ingredients, including the POMs, were ground in the absence of a solvent, after which they were packed with an aluminum foil and heated on a plate at 80 ◦C for only 10 min to obtain PW12@MFM-300(In) composites. The resulting materials exhibited a high crystallinity and stability and no PW12 aggregates were observed on the MOF surface.

**Figure 1.** The hot-pressing synthesis process of PW12@MFM-300(In). Reprinted from [49]. Copyright (2019), with permission from Elsevier.


**5. Conclusions**

A total of 33 mammal species were observed in the study area, including the two rhinoceros species. A variety of mammals were present in the study area, but the basic list of mammals provided here can be expanded if a larger area in the same supersite can be included, more camera traps (especially at the waterholes) and longer survey periods (including seasonal and climatic differences) can be used. On the one granitic catena (hillslope) alone, 23 mammal species were found across all four zones, 21 species at the waterholes and their surrounding areas, 16 species at the granite-boulder outcrops and 13 species at the mud wallows, excluding the two rhino species. Some of these species were similar between different zones and areas. A few species only occurred at the outcrops and waterholes but were not observed on the catena. The hypothesis, that specific mammal species might frequent or associate with certain zone/s on the catena, could not be investigated properly in this current study. This could be due to zones being too small to limit mammals to a specific zone in such an open, natural system. Most mammals also need to pass through certain zones to reach waterholes located outside the catena studied. Some species stay close to the waterhole, while others move larger distances away to feed. The two closer waterholes were less than 4.5 km from the centre of the study area and that is close enough for non-mobile water dependent species to reach, while the furthest one was 6 km away and can even be reached by mobile water-dependent species.

If more than one catena can be included in future studies, it could provide a better understanding of how these mammals use similar zones on other hillslopes in the area and how the various feeding guilds interact with the environment on a landscape scale. Small differences were found in mammal species presence between the three survey periods but the extreme drought possibly limited species richness. The presence of a variety of different sized mammals (small to mega-sized) from different feeding guilds (herbivores to carnivores and omnivores) and dietary classes (grazer to browser and mixed feeder) most probably indicate a functioning ecosystem consisting of various interlinked trophic levels.

Some mammal species may not have been recorded because of the extreme drought during the study period (lack of predator cover and food availability forced them to leave the area), normal movement or migration (they are only present during a certain season or opportunistically arrive when conditions are right—which might have been outside the survey periods) or simply because they travelled outside the view of the cameras in the study area. There is thus scope for future studies to add to the basic list of mammals observed during this study to make it more complete, such as to include a longer time period, a wetter period with normal precipitation, seasonal variation, other types of animals (i.e., birds, reptiles, amphibians, small rodents, invertebrates), etc. It is also known that the numbers of certain mammal species (group sizes) can fluctuate seasonally due to availability of food—in summer they aggregate into large groups, while in winter or when conditions become less favourable; they break up into smaller groups and disperse more widely in search of food. All of these factors and more can contribute to the total variety and species richness of catenas inside the Southern Granite Supersite during different climatic, seasonal or environmental conditions. All the knowledge from this study can be beneficial in monitoring and conservation of species in Kruger National Park.

**Funding:** This research was partially funded by the University of the Free State (UFS) Strategic Research Fund and the NRF Thuthuka Grant (TTK14052267732). The UFS Central Research Fund provided the funds to publish this article.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the University of the Free State Interfaculty Animal Ethics Committee (UFS-AED2019/0121 on 22 August 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** There is no data available from this specific study, but some of the larger multidisciplinary project's data are available at the SANParks Scientific Services repository.

**Acknowledgments:** Jeremy Bolton, camera trapping specialist from Bushcam Consulting is acknowledged for his part in camera trapping procedures and assistance with data processing of some results. Errol Cason from Department of Animal Science, UFS, is thanked for his assistance with Table 3. The multidisciplinary team of the UFS is acknowledged. Staff at Scientific Services in Skukuza and the game guards from SANParks are also thanked for all their assistance.

**Conflicts of Interest:** The author declares no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.


*Soilless Cultivation to Secure the Vegetable Demand of Urban and Peri-Urban Population DOI: http://dx.doi.org/10.5772/intechopen.102695*

alternate technologies, such as soilless culture due to the decline in the availability of arable lands and the problem of soil-borne diseases in soil-based cultivation. Due to better water use efficiency in soilless culture, this particular system of cultivation can also be taken to places where water availability is limited. Presently the hydroponics unit setting up cost is too high because of limited adoption but by acceptance and adoption of more and more growers, the cost of the unit can be brought to affordable levels. Further, this technology is not getting popularity as expected in some of the developing and underdeveloped countries due to various reasons, such as high initial investment and the requirement of skilled manpower. Standardized soilless production technology by the public and private research institutions is very important to popularize and create mass awareness among urban and peri-urban growers. In this direction, ICAR-IIHR is not stopping after standardizing the soilless production technology for vegetables but also putting more and more effort into the spread of this particular technology at the national level. ICAR-IIHR soilless culture production technology has already been popularized through various training programmes, exhibitions, magazines, and media. Many growers have already started adopting IIHR soilless culture technology in the cultivation of vegetables, flowers, and medicinal crops.


*5.4. Number of Nodes Alive*

In Table 7, the average number of live nodes is displayed. As it can be concluded from the Table 7, the average number of live nodes was more than 30% of all node counts which confirmed the maintained network stability as it was proposed by our algorithm. Figures 10 and 11 demonstrate the number of live nodes during simulation. The results showed that HCD had better performance compared to other protocols and increased the number of live nodes during simulation. It was due to the balance between the energy consumption of different nodes as it was proposed by the presented protocol and therefore, could prevent node death.

**Table 7.** The average number of live nodes.

**Figure 10.** The average of the number of alive nodes for scenario 1.

**Figure 11.** The average of the number of alive nodes for scenario 2.


*Article* **The New Human** *Babesia* **sp. FR1 Is a European Member of the** *Babesia* **sp. MO1 Clade**

**Claire Bonsergent 1,\*, Marie-Charlotte de Carné 2 , Nathalie de la Cotte 1 , François Moussel 3 , Véronique Perronne 2 and Laurence Malandrin 1,\***

**Abstract:** In Europe, *Babesia divergens* is responsible for most of the severe cases of human babesiosis. In the present study, we describe a case of babesiosis in a splenectomized patient in France and report a detailed molecular characterization of the etiological agent, named *Babesia* sp. FR1, as well as of closely related *Babesia divergens*, *Babesia capreoli* and *Babesia* sp. MO1-like parasites. The analysis of the conserved 18S rRNA gene was supplemented with the analysis of more discriminant markers involved in the red blood cell invasion process: *rap-1a* (rhoptry-associated-protein 1) and *ama-1* (apical-membrane-antigen 1). The *rap-1a* and *ama-1* phylogenetic analyses were congruent, placing *Babesia* sp. FR1, the new European etiological agent, in the American cluster of *Babesia* sp. MO1-like parasites. Based on two additional markers, our analysis confirms the clear separation of *B. divergens* and *B. capreoli*. *Babesia* sp. MO1-like parasites should also be considered as a separate species, with the rabbit as its natural host, differing from those of *B. divergens* (cattle) and *B. capreoli* (roe deer). The natural host of *Babesia* sp. FR1 remains to be discovered.

**Keywords:** *Babesia divergens*; *Babesia* sp. MO1; *Babesia capreoli*; *rap-1a*; *ama-1*; phylogeny

#### **1. Introduction**

Babesiosis is a tick-borne disease affecting a wide range of vertebrates worldwide. Symptoms of this disease are caused by the intraerythrocytic development of Protozoa of the genus *Babesia*, causing fever, jaundice, hemoglobinuria and anemia, possibly leading to death, depending on the *Babesia* species and the host. About one hundred species of *Babesia* have been described and transmission of the parasite between hosts occurs almost exclusively through Ixodid tick bites [1].

Even though humans are not natural hosts of *Babesia*, human infections caused by several different species of *Babesia* have been reported worldwide. *Babesia microti, B. duncani* (WA1) [2] and to a lesser extent *B. divergens*-like (*Babesia* sp. MO1 clade) [3] have been reported to cause disease in humans in the USA. The most prevalent species is *B. microti* responsible of infections that follow a relatively benign course [4]. In Asia, a few cases have recently been reported, caused by *B. divergens*-, *B. venatorum*- or *B. crassa*-like strains [5–7].

In Europe, the first case of human babesiosis was described in 1957 in Croatia [8,9]. In 1997, a review on human babesiosis in Europe reported 24 cases in splenectomized (20/24) and non-splenectomized (4/24) patients, 46% of which were fatal even in nonsplenectomized patients (2/4) [10]. At that time, the molecular diagnosis of the parasite species was lacking and cases were attributed to *B. divergens* based on morphological and/or serological grounds. A few years later, molecular analysis revealed a new etiological babesiosis agent, *Babesia* sp. EU1, which was found to be responsible for human cases in Austria, Italy [11], Germany [12] and Sweden [13]. Human babesiosis cases due to *B.*

**Citation:** Bonsergent, C.;

de Carné, M.-C.; de la Cotte, N.; Moussel, F.; Perronne, V.; Malandrin, L. The New Human *Babesia* sp. FR1 Is a European Member of the *Babesia* sp. MO1 Clade. *Pathogens* **2021**, *10*, 1433. https://doi.org/10.3390/pathogens 10111433

Academic Editors: Estrella Montero, Jeremy Gray, Cheryl Ann Lobo and Luis Miguel González

Received: 1 October 2021 Accepted: 30 October 2021 Published: 4 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

*microti* have been reported in Europe but usually they are imported cases from the USA [14], with only one autochtonous case reported to date in Germany [15]. Severe sporadic cases are usually attributed to *B. divergens* [16–24]. However, molecular confirmation of the species is not always undertaken [25–27]. Serological analysis and morphology on smears are not sufficient to ascertain *B. divergens* as the etiological agent. Even for specialists, the morphological distinction of *B. divergens* from *Babesia* sp. EU1 on smears is impossible [11,12]. Confirmed cases of babesiosis due to *B. divergens* can remain serologically negative [28–30], and serology can be confusing due to dot-like reactivity patterns of most human positive sera, concentrated at the apical pole of the parasite [31]. This reactivity pattern was confirmed with a serum from a clinically and molecularly confirmed human *B. divergens* case in Finland [18,31].

The phylogenetic group including *B. divergens* gathers different named or as yet unnamed species that are very closely related, and we will refer to this group as *B. divergens*like. *B. divergens* is indeed closely related and can be confused with *B. capreoli*, a parasite frequently found in roe deer in Europe, due to their high 18S rRNA sequence relatedness [32,33]. However, the conservation of three base differences in this gene between isolates of *B. divergens* (pathogen of cattle/humans) and isolates of *B. capreoli* (pathogen of roe deer), linked to different in vitro host ranges, allowed the delineation of these two species [33]. *Babesia* sp. MO1 also belongs to this phylogenetic group, and is responsible for a small number of severe or fatal human babesiosis in splenectomized patients in the USA [3,34–36]. Cottontail rabbits are the natural hosts of *Babesia* sp. MO1 [37,38]. In vitro cultivation features as well as in vivo experimental infections demonstrated the incapacity of this genetic variant to infect cattle, and, combined with 18S rRNA sequence differences, led to its species differentiation from *B. divergens* and the provisional name *Babesia* sp. MO1 [39–41].

In splenectomized patients, babesiosis due to *B. divergens* is fulminant with symptoms that appear within 1–3 weeks post infection, with persistent high fevers and headaches, followed by severe intravascular hemolysis, hemoglobinuria, and jaundice. Babesiosis in splenectomized patients is often fatal in Europe, as diagnosis and therefore adequate treatment are often delayed due to uncharacteristic flu-like symptoms and the infrequency of cases [42,43]. Severe symptoms and fatal cases also occur in non-splenectomized patients with known or unknown predispositions such as splenic dysfunction or a rudimentary spleen [18,19]. In immunocompetent patients, *B. divergens* infection is associated with flu-like symptoms shortly after a tick bite [29] or may remain asymptomatic [44].

In the present study, we describe an unusually mild babesiosis in an asplenic patient in France, originally suspected to be caused by *B. divergens*. Intrigued by the unusual course of infection, we carried out the molecular characterization of the responsible agent. As the 18S rRNA gene is rather conserved within the *B. divergens* taxonomic group, and therefore not sufficiently informative, we supplemented the molecular description with two additional and more variable markers: the apical membrane antigen 1 (*ama-1*) and the rhoptryassociated-protein-1a (*rap-1a*) genes. Molecular characterization and polymorphism of these two genes were also analyzed for different members of the *Babesia divergens*-like phylogenetic group, including the phylogenetically closely related *B. capreoli* and *Babesia* sp. AR1 identical to *Babesia* sp. MO1 but from a patient in Arkansas [35], and compared to available sequences of these genes for *B. divergens*.

#### **2. Results**

#### *2.1. Babesia* sp*. FR1: Report of the Clinical Case*

A 56-year-old man came into the emergency room with a suspected meningitidis syndrome. He was Caucasian and his only notable antecedent was a splenectomy in 2001 following a skiing accident (pneumococcal vaccine administered in November 2016, no *Haemophilus* nor meningococcal vaccines).

The patient stayed on the Île de Ré from 4 August 2017 to 24 August, then from 29 August to 3 September, at a house located at the edge of a forest. He also stayed in Béthune from 25 August to 28 August. He had a fever for 2 weeks associated with headaches. He then developed severe asthenia, sweats, tachypnea, myalgia, and elbow, shoulder, and knee arthralgia.

The first blood test (5 September) revealed thrombopenia: 99 giga/L, CRP 65.6 mg/L, ASAT 82U/L, ALAT 73U/L. On 9 September 2017, he developed vomiting, photophobia and a stiff neck, which led to the patient being transferred to hospital (11 September). Nothing specific was revealed by non-injected brain CT. Lumbar puncture was normal, and culture was sterile. Blood tests revealed the following: platelets 71 giga/L, leukocytes 8.40 giga/L (PNN 7.056 giga/L, lymphocytes 0.670 giga/L), hemoglobin 14.4g/dL, ASAT 57 U/L, ALAT 61 U/L, GGT 128 U/L, PAL 188 U/L, normal kidney function.

On 12 September, when admitted to the infectious disease unit, clinical examination showed fever, asthenia, and non-significant axillary lymph nodes. The same day, a blood smear showed red blood cells with *Babesia* corpuscles inside, reaching a parasitemia of 3.7% (Figure 1). Blood analysis revealed thrombopenia (60 giga/L) and hemolysis signs without anemia (Hb 14.4 g/dL, LDH 890 U/L). Lyme, HCV and HBV serologies were all negative, and protein electrophoresis was normal.

**Figure 1.** Blood smears of *Babesia* sp. FR1 used to diagnose the *Babesia divergens*-like infection of the patient. Human red blood cells infected with dividing pear shaped merozoites are visible, as well as rounded trophozoites. Bar = 5 µm.

Antimicrobial therapy was undertaken on the same day with Atovaquone (750 mg/12h) and Azithromycine (500 mg on day 1 then 250 mg per day). The patient rapidly felt better with apyrexia and disappearance of all symptoms. On 14 September, parasites were still detected on the blood smear and cytolysis was persistent.

Diagnosis of a *B. divergens*-like infection was confirmed by serology (IFAT with *B. divergens* antigen) with a titer of 1:1024 and by PCR on the 18S rRNA gene as described in materials and methods. Sequencing of the amplified 18S rRNA gene portion confirmed that the responsible agent was closely related to *B. divergens*, the most commonly responsible agent of human babesiosis in France, but different.

Control of the patient's infectious status was performed 16 months later. Serology using the same antigen remained positive with a titer reduced to 1:128. PCR was negative.

#### *2.2. Analysis of 18S rRNA Sequences and Position of Babesia* sp*. FR1 within the B. Divergens-Like Phylogenetic Group*

A 1641 bp sequence was obtained for *Babesia* sp. FR1, covering the positions that are discriminant among members of the *B. divergens*-like phylogenetic group: nucleotide positions 631, 663, 819, and 1637. The sequence is highly similar (99.95%) to published *Babesia* sp. MO1-like and *B. capreoli* sequences, with only one nucleotide modification at position 819 and 663 respectively. It is also related to *B. divergens* (99.9%) with two nucleotide substitutions at positions 631 and 1637 (Table 1).

likely vector according to [36]. b corresponding to position described in [33]. c not determined.

> The sequence of 1643 bp from the Arkansas case [35] obtained in this study (named *Babesia* sp. AR1) was 100% identical to the first *Babesia* sp. MO1 case from Missouri (Gen-Bank AY048113) [3], to the Kentucky case (GenBank AY887131) [34] and to the cottontail rabbit isolates [38]. They differ from *B. divergens* by three mutations at positions 631, 819 and 1637 (99.8% identity), and from *B. capreoli* by two mutations at positions 663 and 819 (99.9% identity) (Table 1).

#### *2.3. Major Differences in Ama-1 and Rap-1a Genes within the B. divergens-Like Phylogenetic Group*

Before analyzing the detailed sequence polymorphism of *ama-1* and *rap-1a* genes between the *B. divergens*-like phylogenetic group members, some major differences in gene sequences appeared on the alignment (Figure 2).

Complete *ama-1* sequences (sizes between 1803 and 1857 bp) were obtained in this study for four clonal lines of *B. capreoli*, for *Babesia* sp. FR1 and for *Babesia* sp. AR1, and were compared to *B. divergens ama-1* sequences [45]. An 18 bp sequence located between bases 553 and 570 was absent only in *Babesia* sp. AR1, corresponding probably to a deletion, which did not modify the translation frame (Supplementary Figure S1). *Babesia* sp. FR1 *ama-1* sequence differs from all the others by an insertion of a 36 bp sequence located between bases 1496 and 1531. The inserted sequence is highly similar to an upstream 36 bp sequence (differing by two nucleotides) and seems therefore to correspond to a gene conversion event of this small gene portion.

**Figure 2.** Major differences in *ama-1* and *rap-1a* genes between members of the *B. divergens*-like phylogenetic group. (**a**) Copy number, and presence (Y) or absence (N) of insertion/deletion in *ama-1* and *rap-1a* genes; (**b**) Schematic representation of the major differences and their positions in gene sequence. The colors used in the part (**a**) correspond to the colors used in the graphical representation of the corresponding deletions/insertions in the part (**b**). The deleted or absent regions are dashed.

> Regarding the *rap-1a* gene, partial sequences (sizes between 1203 and 1236 bp) were obtained for four clonal lines of *B. capreoli*, for *Babesia* sp. FR1 and for *Babesia* sp. AR1, and were compared to *B. divergens rap-1a* sequences [46]. Comparison of *rap-1a* sequences from *B. divergens*, *Babesia* sp. AR1, and *Babesia* sp. FR1 highlighted a 3 bp deletion (nucleotides 1012 to 1014) in *Babesia* sp. AR1 only and a 33 bp deletion located between bases 1034 and 1066 in the 12 *rap-1a B. divergens* sequences performed, which did not modify the translation frame. As superposed chromatograms were observed for *B. capreoli* at the 30 end of the *rap-1a* gene, the presence of multiple copies of this gene was suspected and confirmed by cloning/sequencing and subsequent specific amplifications of each gene copy from each of the four isolates. Two *rap-1a* gene copies were observed, which we named *rap-1a1* and *rap-1a2*. The *rap-1a1* copy was characterized by the absence of the two deletions, as found for *Babesia* sp. FR1. The *rap-1a2* copy contained the 33 bp deletion only and therefore resembled *B. divergens rap-1a*.

> These major deletions/insertions were not included in the sequence identity calculations, nor in the phylogenetic analyses, as they represent one-time, usually non-reversible events, with different evolutionary tempo and mode compared to substitutions.

#### *2.4. Intraspecific Sequence Diversity of Rap-1a and Ama-1 within B. divergens and B. capreoli*

The genetic variability within the *B. divergens* and *B. capreoli* strains have been analyzed previously for the 18S rRNA gene and no variations were found [33].

Intraspecific genetic variability of *B. divergens ama-1* and *rap-1a* genes was previously analyzed in studies performed at our lab and was found to be very low [45,46]. Sequence identities higher than 99.5% were highlighted for both genes, when comparing sequences of the same set of nine and twelve French isolates, for *ama-1* and *rap-1a* respectively (Table 2 and Table S1). The *ama-1* sequences showed between 99.9 to 100% conserved sites; two similar nucleotide substitutions were noted in *ama-1* sequences of 1505B F14, 3601B E2 and Rouen87 F5 isolates, compared to *ama-1* sequences of the other six clonal lines. The *rap-1a* sequences showed sequence identities between 99.6 and 100%, corresponding to a pairwise maximum of six nucleotide substitutions.

**Table 2.** Genetic variability of 18S rRNA, *ama-1* and *rap-1a* genes within *B. divergens* and *B. capreoli*.

In the case of *B. capreoli*, we analyzed the genetic polymorphism of *ama-1* and *rap-1a* for four isolates collected and cultivated at our laboratory from previous studies [33,47]. The *ama-1* sequences showed sequence identities between 99.7 to 100%. The 2770 F6 and CVD08 005 *ama-1* sequences were identical and up to nine polymorphic sites were identified resulting in six different nucleotide substitutions between *ama-1* sequences of 2704C and 2801 F10 isolates, and were compared to the other two identical *ama-1* sequences.

The two copies of the *rap-1a* gene (*rap-1a1* and *rap-1a2*) were identified in all four *B. capreoli* isolates. Sequence variability of each gene copy was low (less than seven nucleotide substitutions), and identities ranged between 99.5–100% and between 99.6–100% among the *rap-1a1* and the *rap-1a2* sequences, respectively. Sequence identities between *rap-1a1* and *rap-1a2* copies ranged between 96.1 and 96.5%. Most substitutions specific to each gene copy (39 positions) were non silent (39 substitutions resulting in 28 amino acid modifications), with a majority of substitutions on the first (nine substitutions) and/or second codon position (16 substitutions).

#### *2.5. Genetic Variability within the B. divergens-Like Phylogenetic Group*

As explained above, sequence identities were calculated without the regions corresponding to deletions/insertions and are presented as a contingency table including all three analyzed genes (Table 3). In general, the *ama-1* gene seemed to be more conserved than the *rap-1a* gene as the percentage of sequence identities ranged between 94.3 to 98.7% for *ama-1* and between 86.6 to 98.7% for *rap-1a*. For both genes, the lowest sequence identities were evidenced between *B. divergens* and all other analyzed *Babesia* within the group. The highest sequence identities for *ama-1* and *rap-1a* were obtained between *Babesia* sp. FR1 and *Babesia* sp. AR1 sequences (98.7% identities for both genes). *B. capreoli* was found to be more closely related to *Babesia* sp. AR1 and *Babesia* sp. FR1 than to *B. divergens*.

It was not possible to determine if one of the two copies of *B. capreoli rap-1a* was more related to the unique *rap-1a* gene sequence of other members of the phylogenetic group, as sequence identity values were highly similar.

**Table 3.** Contingency table for 18S rRNA, *rap-1a* (partial cds) and *ama-1* genes. For the 18S rRNA sequences, the number of nucleotide differences between 18S rRNA gene sequences are indicated in red, instead of the identity percentages. For *ama-1* and *rap-1a* genes, percentage of identities are indicated in green and blue, respectively. Sequence identities with each *rap-1a* copy (*rap-1a1* and *rap-1a2*) are indicated. The identities are calculated excluding the deletions and duplication indicated in Figure 2. Accession numbers of sequences used to perform the analysis are indicated in supplementary Table S1.

## *2.6. Phylogenetic Analysis*

The phylogenetic analyses based on 18S rRNA, *ama-1* and *rap-1a* genes were concordant and confirmed the placement of the *Babesia* sp. FR1 into the *B. divergens*-like phylogenetic group, with strong bootstrap values of 100 (Figures 3–5). According to the 18S rRNA phylogenetic analysis, and despite the high level of conservation of this marker, two sister groups were supported by good bootstrap values, and *Babesia* sp. FR1 clustered with *B. capreoli*, *Babesia* sp. AR1 and *Babesia* sp. MO1 (bootstrap of 73), and not with *B. divergens* (forming the second cluster supported by a bootstrap value of 89) (Figure 3). The separation of these two clusters was also well supported in the phylogenetic analysis with *ama-1* and *rap-1a* as markers (Figures 4 and 5). The *B. divergens* clade was supported by bootstrap values of 99 and 100 (*ama-1* and *rap-1a* respectively). The *B. capreoli*/*Babesia* sp. AR1/*Babesia* sp. FR1 clade was also well supported by bootstrap values of 100 (*ama-1* and *rap-1a*), but splits on the one hand into a subclade with *B. capreoli* (bootstraps of 100 and 83) and on the other hand into a second subclade with *Babesia* sp. FR1 and *Babesia* sp. AR1 (bootstraps of 100 and 99). The two *Babesia capreoli rap-1a* copies clustered into two sister groups with strong support (100).

**Figure 3.** Maximum likelihood unrooted phylogenetic tree of *Babesia* from the *Babesia divergens*-like phylogenetic group based on partial 18S rRNA sequences (1189 bp in the final data set). Branch support/bootstrap values are indicated at each node. *Babesia* sp. FR1 sequence obtained in this study is emphasized in red. (**a**) Scale bar indicates nucleotide substitution rate per site. (**b**) Topology of the tree allowing a better visualization of the bootstrap values; hosts of *Babesia* isolates are indicated.

**Figure 4.** Maximum likelihood unrooted phylogenetic tree of *Babesia* from the *Babesia divergens*-like phylogenetic group based on partial *ama-1* gene sequences (1728 bp in the final data set). Branch support/bootstrap values are indicated at each node. *Babesia* sp. FR1 sequence obtained in this study is emphasized in red. (**a**) Scale bar indicates nucleotide substitution rate per site. (**b**) Topology of the tree allowing a better visualization of the bootstrap values.

**Figure 5.** Maximum likelihood unrooted phylogenetic tree of *Babesia* from the *Babesia divergens*-like phylogenetic group based on partial *rap-1a* gene sequences (1138 bp in the final data set). Branch support/bootstrap values are indicated at each node. *Babesia* sp. FR1 sequence obtained in this study is emphasized in red. Scale bar indicates nucleotide substitution rate per site.

#### **3. Discussion**

Most human babesiosis cases are recorded in North America and are mainly due to *Babesia microti*, sporadically to *B. duncani* (*Babesia* sp. WA1) and to *Babesia* sp. MO1-like parasites. Sporadic cases were reported in Asia, Africa, and South America, with diverse and often partially characterized etiological agents [48]. In Europe, human babesiosis is rare and *B. divergens* is the main causal agent [43,49]. The most impacted countries are France, Ireland and Great Britain, and in France, Western regions and Normandy are most affected [10], due to substantial farming of bovines, the natural host of *B. divergens* [50].

The patient was most probably bitten by a tick on the Île de Ré, even if he had no recollection of a tick bite. This is the most probable place of tick acquisition by the patient, as it is close to a forest, where the abundance of the potential vector *I. ricinus* is high, increasing the risk of contracting tick-borne pathogens [51]. The patient was asplenic, which is also a major risk factor for severe or fatal babesiosis [10,43,49]. However, the symptoms in this patient developed slowly (two to three weeks between the onset of symptoms and admission to hospital) despite aspleny, while *B. divergens'* course of infection in such cases is usually fulminant [42]. Biological diagnosis of the provisionally named *Babesia* sp. FR1 was based on a blood smear, which led to the administration of antibabesial therapy (Atovaquone and Azithromycine) as soon as practicable. This treatment was effective, as symptoms rapidly disappeared, and parasite clearance was attested 16 months later by a negative PCR, correlated with a reduction of the serology titer.

Molecular characterization of *Babesia* sp. FR1 required a deeper analysis. Sequence and phylogenetic analysis of 18S rRNA revealed that it was genetically close but different from typical *B. divergens* isolates infecting cattle or humans (two polymorphic sites at positions 831 and 1637) [33], but that it closely resembled the American *B. divergens*-like parasite *Babesia* sp. MO1. Despite the genetic difference with *B. divergens*, the infection could be diagnosed using *B. divergens*-specific serological tools (IFAT), confirming anyway a close relationship with *B. divergens.*

We decided to explore new markers to improve knowledge on the *B. divergens*-like phylogenetic clade and to correctly position this new isolate within this species complex. We chose *rap-1a* and *ama-1* for two reasons. First, both genes code for proteins involved in the process of red blood cell invasion by the parasite [52], and as host range/specificity is an important biological feature in the description of this intra-erythrocytic obligatory parasite, they represent markers of interest. Second, we know from previous studies that both genes were well conserved among *B. divergens* isolates from cattle or humans [45,46]. Their interspecies divergence remained to be determined.

Regardless of the marker used, the sequences of *B. divergens* (cattle as a natural host) are grouped in a cluster well-separated from the other two clusters corresponding to *B. capreoli* and *Babesia* sp. MO1/AR1/FR1. Phylogenies based on more discriminant markers (*rap-1a* or *ama-1*) placed *Babesia* sp. FR1 in the cluster formed by isolates responsible for cases of human babesiosis in the USA represented by *Babesia* sp. AR1. This cluster is separated from the cluster of *B. capreoli* sequences, and from the cluster of *B. divergens* sequences. We can therefore conclude that *Babesia* sp. FR1 is not a *B. divergens*.

The phylogenetic group containing *Babesia* sp. MO1 is sometimes referred to as the *B. divergens* US lineage [53,54]. However, *Babesia* sp. FR1, which clusters with *Babesia* sp. MO1 and *Babesia* sp. AR1, was clearly acquired locally and is an autochthonous case as the patient did not travel to the USA in the months before the onset of the symptoms. Therefore, we not only confirm in the present study that *Babesia* sp. MO1-like sequences form a well-supported taxon, but we also highlight that the geographical distribution of this group is not restricted to the USA, but extends to Europe as it includes *Babesia* sp. FR1. The three clusters within *Babesia divergens*-like, i.e., *B. divergens*, *B. capreoli,* and *Babesia* sp. MO1 like might be associated with their natural host rather than with geographic distribution. Humans are only incidental hosts for parasites belonging to the *B. divergens*-like group, the natural hosts being cattle for *B. divergens*, roe deer for *B. capreoli* and rabbits for *Babesia* sp. MO1. The natural host for *Babesia* sp. FR1 has not been characterized but could also well be a Laporidae, especially in the Île de Ré context, a highly touristic and populated island where cattle and cervids are rare or absent, due to limited forested areas dominated by resinous trees (mainly maritime pine trees) and typical local productions (vineyards and salt marshes). The European rabbit (*Oryctolagus cuniculus*) is highly abundant on this island where it has been pullulating since the 2000s, and could therefore be the potential natural host for *Babesia* sp. FR1.

In this study, we did not include isolates described as *B. divergens* in sika deer described in Japan or in humans in China [6,53,54]. Our goal in this study was to characterize and correctly place the new *Babesia* sp. FR1 isolate in the phylogenetic group of *Babesia divergens*like, among biologically well-characterized isolates, i.e., whose host range has been studied and whose parasites have been cultured [33,38–41,55]. The isolates described in Japan from sika deer and named *B. divergens* are not included in the *B. divergens*-like phylogenetic group because they differ at the 18S rRNA sequence from all other members of this group by at least six conserved substitutions all of which are different from those described within this group. These isolates form a sister group to *B. divergens*-like. The name *B. divergens* should be reserved for isolates from cattle or humans whose 18S rRNA sequences match the many descriptions already published [18–23,29,30,33,49]. There is no evidence that isolates

from sika deer are capable of infecting either cattle, gerbils (*B. divergens* experimental host), or humans.

The sequences named as "*B. divergens*" and described in humans in China [6] actually match those described for *B. capreoli*, with the characteristic differences at positions 631 and 663 [33]. This information raises the possibility of human infections by *B. capreoli*, a species that has never been molecularly characterized as responsible for symptomatic cases of human babesiosis in Europe and described as not growing in vitro in human red blood cells [33]. However, short-term asymptomatic carriage of parasites could occur in humans in geographic areas with high parasite and vector prevalences. We unsuccessfully attempted to obtain DNA from these parasites to include them in our study.

In the present study, we characterized *rap-1a* genes in the *B. divergens*-like phylogenetic group. The *rap-1a* genes belong to a multigene family, and multiple copies have already been demonstrated in a few *Babesia* species: two copies in *B. bovis* and *B. canis*, four to five in *B. ovis*, at least seven copies in *Babesia* sp. Xinjiang, eleven in *B. bigemina* and twelve in the *B. motasi*-like group members [52,56–61]. The multiple copies of *rap-1a* are usually different, allowing their differentiation, except in the case of *B. divergens* where the presence of two identical copies was highlighted when its genome was sequenced [52]. It is highly probable that two identical and therefore undistinguishable copies of *rap-1a* exist in all the *B. divergens* isolates characterized. We cannot exclude the presence of two identical copies also for members of the *Babesia* sp. MO1-like clade. In all *B. capreoli* isolates analyzed, two different but closely related *rap-1a* copies, named *rap-1a1* and *rap-1a2* (sequence identities of about 96%) were identified. Each copy is equally different from either *B. divergens*, *Babesia* sp. FR1 or *Babesia* sp. AR1 *rap-1a* genes, and the two copies place as sister groups to each other, indicating a gene duplication that occurred after *B. capreoli* speciation. While genetic divergence occurred between the two *rap-1a* copies of *B. capreoli*, it was not the case between *B. divergens rap-1a* copies. *B. divergens rap-1a* gene has been previously characterized and its genetic variability among cattle and human isolates was found to be limited [51,62,63]. A greater sequence diversity among *B. capreoli* isolates compared to *B. divergens* was also highlighted in the case of the merozoite surface antigen *Bc37/41* compared to *Bd37*, and a greater selection pressure was hypothesized [64]. This could also explain the sequence divergence between the two copies of *rap-1a* in *B. capreoli* and not between the two *B. divergens* copies of *rap-1*. But a more recent event of the *rap-1a* gene duplication in *B. divergens* could also explain the difference in sequence divergence between the two copies. Whether the last common ancestor of members of the *B. divergens*-like group possesses one or two copies of *rap-1a*, or when and how many times *rap-1a* gene duplication occurred in the speciation process is difficult to evaluate. Despite the presence of all motives that characterized RAP-1 family members, *rap-1b* sequence identity with the other two copies of *rap-1a* in the *B. divergens* genome is extremely low (45%) and was not amplified with the primers used.

In conclusion, we describe here a case of human babesiosis in Europe (France) due to a *Babesia* isolate more closely related to the American *Babesia* sp. MO1 and AR1 than to *B. divergens*. Using two discriminant molecular markers, our study confirms the existence of three phylogenetic clades within the *B. divergens*-like group that would deserve the rank of species as their phylogenetic classification corresponds with their natural hosts; *B. divergens* natural host is cattle, *B. capreoli* infects mainly roe deer, and *Babesia* sp. MO1-like parasites probably infect Laporidae.

In Europe, diagnosis of human babesiosis is complicated due to its infrequency which often leads to a delayed detection and treatment. This delayed treatment promotes the development of a fulminant manifestation of this parasitic disease, in particular for asplenic or immunocompromised patients. Thus, the infection results often in the death of the patient, which could probably have been avoided by a more precocious diagnosis and treatment [23,26]. In Europe, the serological and molecular tools developed to diagnose *B. divergens* infections should principally be adequate to detect *Babesia* sp. FR1 infections. However, it needs to be taken into account that atypical immunofluorescence patterns (dots

and weak fluorescence of the parasite surface) may lead to a negative conclusion when carrying out an immunofluorescence test. Prophylactic treatments are advised, such as wearing long clothes and performing skin examination for tick detection after exposure to high-risk environments.

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

#### *4.1. Babesia Isolates and DNA Origins*

A preliminary identification of *Babesia* sp. FR1 responsible for the mild form of babesiosis was performed on blood smears stained with May-Grünwald Giemsa. Further diagnosis was carried out by serology (IFAT with *B. divergens* antigen) [31], as well as 18S rRNA gene amplification [33] and sequencing from blood DNA extracted using the Nucleospin Blood kit according to the manufacturer's instructions (Macherey-Nagel, Düren, Germany).

*B. capreoli* isolates were collected and characterized in previous studies performed at our lab [33,47]. We included in our analysis four in vitro cultivated isolates from roe deer blood samples or spleen, from three different regions of France (Supplementary Table S1). *B. divergens* isolates were also cultivated in vitro from acute piroplasmosis cases in cows (11 isolates) or in humans (one isolate) [55].

DNA from cultured *B. capreoli* clonal lines (2704C, 2770 F6, 2801 F10 and CVD08 005) was extracted as previously mentioned.

DNA from one American case of human babesiosis was kindly provided by Mayo Medical Laboratory, Rochester, USA. This fatal case occurred in an asplenic patient, in Arkansas in 2015, with a possible acquisition through transfusion [35]. Due to its geographical origin, we named this isolate *Babesia* sp. AR1. It was characterized as a *Babesia* sp. MO1-like babesiosis etiological agent.

As all these isolates are very closely related to *B. divergens*, we have used throughout the manuscript the terminology *B. divergens*-like phylogenetic group to qualify the following isolates, species or clades: *B. divergens*, *B. capreoli*, *Babesia* sp. MO1, *Babesia* sp. AR1, and the new *Babesia* isolate, named *Babesia* sp. FR1.

#### *4.2. Comparison of 18S rDNA Sequences within the B. divergens Taxonomic Group*

*Babesia* sp. AR1 18S rRNA sequences (560 bp) from the previously mentioned isolates were kindly provided by Mayo Medical Laboratory, Rochester, USA. Published *B. capreoli* as well as *B. divergens* 18S rRNA sequences were used as a comparison [33]. Their origin and accession numbers are described in Supplementary Table S1. The partial 18S rRNA sequence of *Babesia* sp. FR1 was obtained using the same primers [33] (Table 4). With the aim of obtaining sequences of comparable sizes, the 18S rRNA partial sequence of *Babesia* sp. AR1 was also amplified with these primers and sequenced. The alignment was done using the ClustalW program as implemented in the Geneious R6 software (https://www.geneious.com accessed on 1 October 2021).

#### *4.3. Amplification of Ama-1 (Apical Membrane Antigen-1) and Rap-1a (Rhoptry Associated protein-1) Genes for B. capreoli, Babesia* sp*. AR1, and Babesia* sp*. FR1*

PCR was performed to amplify the *ama-1* and *rap-1a* genes of *B. capreoli*, *Babesia sp.* AR1, and *Babesia* sp. FR1 isolates using ama1-S1/ama1-R3 and rap1-fw/rap1-rev primers respectively (Table 4). Reactions were carried out in 30 µL reaction mixtures containing 1 X GoTaq buffer, 4 mM MgCl2, 0.2 mM of each dNTP (Eurobio Scientific, Les Ulis, France), 1 unit GoTaq G2 Flexi DNA Polymerase (Promega, Madison, WI, USA), 0.5 µM of each primer and 1 µL of DNA template. The amplification conditions comprised 5 min at 95 ◦C followed by 40 cycles of 30 s at 95 ◦C, 30 s at the temperatures indicated in Table 4, 1 min 30 s at 72 ◦C, and a final extension at 72 ◦C for 5 min. The amplified fragments were purified with the ExoSAP-IT reagent according to the manufacturer's instructions (Affymetrix, Santa Clara, CA, USA) and sequencing was performed on both strands (Eurofins Genomics, Ebersberg, Germany) using the same primers for *rap-1a* gene, or using primers distributed

## along the sequence for *ama-1* gene (Table 4). Sequences were then assembled using the Geneious R6 software.

**Table 4.** Description of the primers used in this study for gene amplification as well as sequencing.

For the four isolates of *B. capreoli* (2704C, 2770 F6, 2801 F10 and CVD08 005), preliminary sequencing results of *rap-1a* gene highlighted superposed chromatograms at the gene 3 0 end, suggesting the presence of multiple copies of this gene, a frequent feature for this gene. The PCR products were therefore cloned in the pGEM-T easy vector according to the manufacturer's instructions (Promega, Madison, WI, USA), to determine the number and sequences of the putative different copies of the *rap-1a* gene. *Escherichia coli* strain BL21 was transformed with the plasmid constructions and colonies with the expected inserts were selected by direct colony PCR using vector primers: T7 and SP6. Recombinant plasmids were then isolated using the Nucleospin Plasmid kit (Macherey-Nagel, Düren, Germany) and both strands of the inserts were sequenced using vector primers (Eurofins, Genomics, Ebersberg, Germany). Then primers were designed to selectively amplify the different *rap-1a* gene copies of *B. capreoli* isolates (Table 4). Primers rap1-a1-fw or rap1-a2-fw were associated with primer rap1-rev to amplify the 30 part of respective *rap-1a* copies. Primers rap1-a1-rev or rap1-a2-rev were associated with primer rap1-fw to amplify the 50 part of *rap-1a* copies (same reaction and cycling conditions as above). PCR products were purified and sequenced as already mentioned.

#### *4.4. Comparison of Ama-1 and Rap-1a DNA Sequences for B. divergens-Like Phylogenetic Group Members*

The resulting *ama-1* and *rap-1a* DNA sequences of *B. capreoli*, *Babesia* sp. AR1 and *Babesia* sp. FR1 were aligned along with the published *ama-1* and *rap-1a* DNA sequences of *B. divergens* isolates using the ClustalW program as implemented in the Geneious R6 software.

#### *4.5. Phylogenetic Analysis*

Phylogenetic relationships within the *Babesia divergens*-like group were inferred using published sequences available in GenBank (Supplementary Table S1) and sequence data produced in the present study (18S rRNA, *ama-1* and *rap-1a* sequences). Sequences were aligned using Muscle as implemented in MEGA version X [65]. Phylogenetic analyses used a trimmed alignment of 1189 bp with complete deletion option for the 18S rRNA gene, 1728 bp and 1138 bp with complete deletion option for the *ama-1* and *rap-1a* coding sequences respectively. Maximum likelihood phylogenetic trees were produced using MEGA-X, with 1000 bootstrap replications based on the Tamura 3-parameter model [66] for the 18S rRNA gene and the *ama-1* gene, and based on the Kimura 2-parameter model [67] model for *rap-1a* gene. For the *ama-1* and *rap-1a* sequences, the three codon positions were included. The appropriate model of nucleotide substitution for ML analysis was selected based on the Bayesian Information Criterion (BIC) computed by MEGA-X.

#### *4.6. Genbank Deposition*

Nucleotidic sequences obtained in this study were submitted to GenBank with accession numbers MZ825347 and OK086051 for the 18S rRNA sequence of *Babesia* sp. FR1 and *Babesia* sp. AR1 respectively, MZ836259 and MZ836261 for the *ama-1* sequences of *Babesia* sp. AR1 and *Babesia* sp. FR1 respectively, MZ836258 and MZ836260 for the *rap-1a* sequences of *Babesia* sp. AR1 and *Babesia* sp. FR1 respectively. Accession numbers for *rap-1a* and *ama-1* sequences of *B. capreoli* are presented in Supplementary Table S1.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pathogens10111433/s1, Table S1: Description of *B. divergens* (A) and *B. capreoli* (B) clonal lines used in the study and Genbank accession numbers. Figure S1: (A) Major differences in *ama-1* gene and AMA-1 protein sequences. (B) Major differences in *rap-1a* gene and RAP-1A protein sequences.

**Author Contributions:** Conceptualization, L.M. and M.-C.d.C.; methodology, C.B. and F.M.; validation, C.B., N.d.l.C. and L.M.; formal analysis, C.B.; investigation, C.B., V.P. and M.-C.d.C.; resources, V.P., F.M., M.-C.d.C. and L.M.; data curation, C.B.; writing—original draft preparation, C.B., M.-C.d.C. and L.M.; writing—review and editing, C.B., M.-C.d.C., N.d.l.C., F.M. and V.P..; visualization, F.M., C.B. and L.M.; supervision, L.M.; project administration, L.M.; funding acquisition, C.B., N.d.l.C. and L.M. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Ethical review and approval were waived for this study, because this case report did not involve human subject research.

**Informed Consent Statement:** Written informed consent has been obtained from the patient to publish this paper.

**Acknowledgments:** The authors wish to thank M. Jouglin for technical support.

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

#### **References**


**Control the Metal Grain Boundary Recrystallization Evolution by the Laser Radiation Electric Field Strength Direction Under Cyclic Thermal Loading Control the Metal Grain Boundary Recrystallization Evolution by the Laser Radiation Electric Field Strength Direction Under Cyclic Thermal Loading**

Makin Vladimir Sergeevich Makin Vladimir Sergeevich

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66248

#### **Abstract**

The spatial grating formation at metal surface under the linear polarized laser radiation action is briefly considered. The spatial grating periods are well described in framework of universal polariton model (UPM) and are well-defined physical quantities. The production of new-type gratings (quasi-gratings) at laser power densities lower than the metal melting threshold with power-dependent typical spatial scale and polarization-dependent orientation are discovered. The regularities of quasi-grating production are experimentally studied. The physical model of quasi-grating formation explaining the anisotropic character of metal recrystallization is suggested. The anisotropy is caused by the directed electron flux interaction with grain boundaries. The electron flux results from the drag effect of electrons by surface plasmon polaritons (SPPs). SPPs are excited by incident laser radiation on the surface irregularities including the grain boundaries. The volume analog of considered effect is the electroplastic one, and some of its regularities are considered.

**Keywords:** metal, anisotropic recrystallization, laser radiation, linear polarization, grain boundary movement, surface plasmon polaritons, electrons drag by surface plasmon polaritons, electroplastic effect


1. Introduction

Global population is still increasing, and therefore, more resources are required to fulfill people needs. The demand of energy is growing due to the population increase but also because the presence of new activities such as social networking. Nowadays, most of the vehicles utilize fossil fuels. The fossil fuels are non-renewable resources and they contribute significantly to the global pollution.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Biofuels are a propitious alternative to the fossil fuels reliance, due to their sustainable production based on using biomass as a raw material. Biomass is an organic matter derived from living or recently living beings. The production of biofuels also has some advantages from those produced from non-sustainable resources, for instance, the reduction of greenhouse emissions (GHG) such as the CO2. Another benefit of using biofuels is the development of an agricultural industry and the creation of rural jobs to produce and deliver biomass to biorefineries. Bioethanol is one type of biofuel that can be produced from organic resources such as corn stover, miscanthus, and switchgrass, among others. Bioethanol has several applications in a wide variety of industries. According to government dependencies such as the Energy Information Administration (EIA), an increment of biofuels is expected in the U.S. within the coming years as product of the Renewable Fuel Standard [1] that requires more production of fuel utilizing renewable resources (e.g., 16 billion gallons for 2022).

specific batch of biomass will be less than expected. With a shortage of biofuel, acquiring the slack from a third-party supplier to cover the demand could lead the producer to an increment

Modeling and Optimization of Quality Variability for Decision Support Systems in Biofuel Production

http://dx.doi.org/10.5772/intechopen.73111

41

Biomass has many physical and chemical properties that need to be considered in order to optimize an objective such as minimization of the total cost or maximization of the profit. This chapter introduces a principal component analysis (PCA) to identify significant factors that affect the design and implementation of logistic processes and infrastructure due to the physical and chemical properties of the biomass. Moreover, the chapter presents a two-stage optimization model that take into consideration quality-related costs in order to set up the biorefinery locations and the flows of biomass from the supplier to the producer. The optimization model can be incorporated into a decision supported system (DSS) to solve several

This analysis focuses on LCB [specifically, on switchgrass (Panicum virgatum)], which is a feedstock derived from organic matter that is mainly composed of cellulose and hemicellulose. Examples of LCB are corn stover, wheat straw and switchgrass and there are many activities involved in the production and distribution of this kind of biomass such as harvesting, extracting, packaging, transporting, handling, among others. There are factors in the aforementioned activities that affect the physical and chemical properties of the biomass, and thus, their qualityrelated costs. The consideration of those factors has an effect in the design and implementation

An example of the relationship between the biomass properties and the SC design is the cellulose and hemicellulose (carbohydrates) contents in the LCB. The cellulose and hemicellulose content in the LCB is directly related to the biofuel produced in the conversion process since the carbohydrates are the main component to produce the energy. The more carbohydrates contained in the biomass, the more liters of biofuel obtained from that particular batch of feedstock. Hence, activities that affect the carbohydrate contents need to be improved to

Densification is one of the processes in the SC of biofuel production that affects some of the properties within the biomass. The densification of biomass consists in conglomerating the organic matter in the form of compact structures such as briquettes and pellets, to improve their handling, storage and transportation but also to reduce the level of dry matter loss (DML) in the feedstock. The DML is directly related with the loss of carbohydrates in the biomass. The less organic matter in the batch, the less amount of cellulose and hemicellulose to produce biofuels. Densification also affects other properties in the biomass such as the moisture content, unit density, durability index, as well as other properties specified for the conversion process according to the implemented technology. Controlling the physical and chemical properties under the specification requirements is vital for an efficient conversion. Delivering

instances of interest and aid the decision-making process.

2. A PCA in a switchgrass composition

minimize the impact on the conversion process.

in the overall cost.

of the supply chain (SC).

Biofuels are classified according to the raw material utilized to produce them: (1) the firstgeneration is related to biofuels produced from edible biomass that can be generally used for human consumption (e.g., corn, sugarcane, sugar beet, among others), (2) the secondgeneration are biofuels generated from a wide range of feedstock, including lignocellulosic biomass (LCB), such as perennial grasses, soft and hard wood, up to municipal solid waste, and (3) the third-generation commonly refers to biofuels produced from algal biomass [2]. Biofuels produced from LCB are a feasible option in the U.S. for the coming years as they utilize non-food feedstock and can be grown in marginal lands or are byproducts produced from the wood industry.

Biofuel production requires improvements in strategic areas such as conversion technologies, genetic manipulation of feedstock breeds, and supply chain management of biomass from harvesting areas to conversion facilities, among others in order to become a plausible alternative to fossil fuel production. Supply chain (SC) improvement represents an important area of opportunity in biofuel production due to the fact that environmental, geographical and economic factors are related to operations like harvesting, handling, storing as well as transportation, which have shown significant impact in biofuel yields/cost. Studies have demonstrated that some factors such as storage time affects the physical and chemical properties of the biomass [3], therefore, biomass properties have an important role in the design of the operations required for the production and distribution of biofuel [4].

There are many properties that affect the conversion efficiency depending on the type of conversion technology that is being utilized for the production of biofuels. Moisture is a property of biomass that affects both the thermochemical and biochemical conversion technologies and a drying process that diminishes the moisture content in the biomass up to the required level of humidity is needed. The drying process to meet the specification for the selected conversion technology incurs in a cost that could be reduced/controlled with the implementation of logistics processes and infrastructure design that consider the level of humidity in the feedstock. The utilization of the biomass without meeting the expected specifications could lead the production of biofuels to an inefficient conversion process. Another example of the importance of the biomass properties are the carbohydrates. If the level of carbohydrates does not meet the specification, then, the amount of biofuel derived from that specific batch of biomass will be less than expected. With a shortage of biofuel, acquiring the slack from a third-party supplier to cover the demand could lead the producer to an increment in the overall cost.

Biomass has many physical and chemical properties that need to be considered in order to optimize an objective such as minimization of the total cost or maximization of the profit. This chapter introduces a principal component analysis (PCA) to identify significant factors that affect the design and implementation of logistic processes and infrastructure due to the physical and chemical properties of the biomass. Moreover, the chapter presents a two-stage optimization model that take into consideration quality-related costs in order to set up the biorefinery locations and the flows of biomass from the supplier to the producer. The optimization model can be incorporated into a decision supported system (DSS) to solve several instances of interest and aid the decision-making process.