**1. Introduction**

Solar energy is a renewable energy source capable of addressing environmental concerns and concerns about energy sustainability. The best known and most widely used technology in renewable energy generation is the use of photovoltaic (PV) modules that convert solar energy into electrical energy through photovoltaic cells. Admittedly, much of the solar energy absorbed by PV cells causes an increase in the temperature of the PV module, which causes a reduction in power output and energy efficiency, and negatively affects its performance and lifetime [1]. In addition, the most common climatic factors that influence the conversion efficiency of PV modules are solar radiation [2], ambient temperature and module surface temperature [1], relative humidity, wind speed and accumulated dust or shading problems on the module surface [3].

In recent years, researchers have conducted many reviews and comparative analyses on various proposed cooling techniques [4,5], of which water cooling systems stand out as a good solution to improve electrical efficiency and decrease the degradation rate of PV cells. On the other hand, Vivar et al., 2010 [6], proposed a novel technology called SolWat, which was later proposed [7,8] to improve environmental sustainability (scarcity, water stress and deterioration of water quality) and reduce the energy consumption of a wastewater treatment plant (WWTP) by implementing this technology as a tertiary treatment. This proposal would be very beneficial since energy consumption in the WWTP is a critical

**Citation:** Torres López, J.; Vivar García, M.; Fuentes Conde, M.; Palacios Villa, A.M. Open SolWat System with Cooling of the Secondary Wastewater Effluent from a WWTP on the Front Surface of the Photovoltaic Module for efficient Energy Generation and Reclaimed Water Production. *Environ. Sci. Proc.* **2023**, *25*, 81. https://doi.org/ 10.3390/ECWS-7-14321

Academic Editor: Athanasios Loukas

Published: 3 April 2023

**Copyright:** © 2023 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/).

part of its operation and maintenance costs, especially for tertiary treatments that require high energy consumption, although as they are not required by law, they are not usually included in the wastewater treatment line. In view of these problems, possible solutions can be found in the use of renewable treatment plants [9,10] (to obtain energy) that also allow water to be reused.

2 of 8

The results obtained by Vivar et al., 2021 [7], and Torres et al., 2022 [8], managed to obtain reclaimed water from the secondary effluent of wastewater from a WWTP, after SODIS disinfection treatment [11] under real sunlight for 4 h, and to produce energy simultaneously. Now, this study proposes a new design in SolWat technology, the Open SolWat system. Following the objectives already proposed in previous research, this work aims to: (a) improve the final quality of reclaimed water for reuse and for final discharge to the environment; and (b) obtain energy, but this time the new prototype will significantly increase the energy efficiency in the SolWat PV module by means of a cooling system based on a thin film of water on the front surface of the module.

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

The SolWat technology is based on a solar–photovoltaic hybrid system (water disinfection reactor coupled to a photovoltaic module) that uses only solar energy to purify water and generate electricity. This technology receives solar radiation and uses its broad electromagnetic spectrum for (1) water disinfection thanks to the germicidal effect of ultraviolet (UV) radiation and the thermal effect of far infrared radiation (FIR) that drastically reduce the amount of pathogenic microorganisms present in the water, and (2) electricity production thanks to the visible (VIS) and near infrared (NIR) radiation reaching the photovoltaic module.

The new prototype is called the "Open SolWat system" (Figure 1) and has a dynamic operation mode (with water recirculation). The system consists of a PV module through which a thin water film (1 mm) circulates, allowing cooling of the PV module and solar disinfection of the water, from the top of the module until it is collected in an open water tank, which is exposed to solar radiation and therefore also acts as a solar water disinfection reactor. A reference PV module was also used experimentally as a control system.

**Figure 1.** (**a**) Open SolWat system and reference photovoltaic module. (**b**) Use of the electromagnetic spectrum in Open SolWat.

The PV modules were polycrystalline silicon cells (PV LOGIC, Bredon, UK) consisting of 36 solar cells connected in series with dimensions of 634 × 535 × 25 mm (0.251 m2 cell area) and a nominal power of 45 Wp. This prototype has two water reactors: (a) a reactor formed only by an L-shaped aluminium profile at the top of the photovoltaic module, where the perforated pipe with 53 water outlet microtubes (3 mm water inlet and 2 mm

water outlet) with a separation of 1 cm between them is attached; and (b) the open and translucent water tank (62 × 45 × 18 cm). Water recirculation in the SolWat was performed by a pumping system, with a Xylem Flojet magnetic coupling water pump (NDP14/2, supply voltage: 230 V, input power: 10 W) through a set of pipes that propelled the water sample from the open water tank to the SolWat, with an average flow rate of 8.9 L/min. The water pump was primed with 200 mL of the experimental sample. Experimental tests with 4.2 and 6.2 L volumes were carried out with wastewater and purified and ultrapure (Milli-Q) water samples to study the influence of water flow and turbidity on PV performance. The homogenisation of the samples was achieved thanks to the drop of the water sheet into the open tank and the water pipe with 7 orifices distributed inside the tank.


**1.6.2. Turneaureite**

difference enables chlorapatite to accept smaller cations without the distortion will not be

Svabite is rare accessory mineral in calc-silicate skarns and arsenate analogue of fluorapatite [15] with the composition given by the formula Ca10(AsO4)6F2 (calcium fluorarsenate, CAAP). The mineral was named in 1981 by SJÖGREN [159] according to the Swedish chemist ANTON VON SWAB (1703–1768), who distilled zinc from calamine (smithsonite, ZnCO3 [160]) [161]. Svabite has the same crystal habit as apatite, with rough hexagonal prisms, some of which show rounded pyramidal termination [162]. The structure of mineral svabite is shown in **Fig. 23**.

**Fig. 23.** The structure (perspective view along the *c*-axis) and examples of crystal habits of svabite.

It is brittle mineral that can be white, gray, gray-green, or colorless and transparent. The mineral is considered to be the intermediate between the apatite and the pyromorphite series. Svabite has the average density of 3.7 g·cm−3, and the hardness on the Mohs scale ranges from 4

attempted.

**1.6.1. Svabite**

**1.6. Other members of the apatite group**

36 Apatites and their Synthetic Analogues - Synthesis, Structure, Properties and Applications

**Fig. 24.** Known localities for the mineral svabite.

Turneaureite (Ca5(AsO4)3Cl, calcium chloroarsenate [7],[163]) is hexagonal mineral with the space group P63/M with following crystallographic parameters *a* = 9.81 and *c* = 6.868 Å, *a*:*c* = 1:0.700, *V* = 572.4 Å3 and *Z* = 2. The mineral is Cl analogue of svabite (**Section 1.6.1**) and OH analogue of johnbaumite (**Section 1.6.3**). The structure of turneaureite is shown in **Fig. 25**.

**Fig. 25.** The structure of turneaureite (perspective view along the *c*-axis) and examples of crystal habits of turneaureite.

The mineral occurs47 in Franklin, New Jersey; Längban, Sweden (holotype48); and Balmat, New York [164]. The name honors Dr. FREDERICK STEWART TURNEAURE, Professor Emeritus at the University of Michigan, in recognition of his contributions to the geology and mineralogy of ore deposits. The mineral occurring occasionally as colorless, slightly turbid, prismatic (only the forms {1010} and {0001} are present) crystals up to 1.5 mm long. The mineral is colorless with a vitreous to slightly greasy luster. The hardness on the Mohs scale is 5. Measured and calculated density of mineral is 3.60 and 3.63 g·cm−3, respectively [163], [164].


**Abstract**

In all stages of wound healing, macrophages play a pivotal role by coordinating the repair steps in a timely and accurate fashion. The successful completion of wound healing requires proper spatiotemporal presence and function of macrophages. Diabetes significantly alters the proliferation, polarization and functionality of macrophages, leading to a suboptimal but prolonged pro-inflammatory M1-like phenotype in wound macrophages and a failure of their late transition to a reparative M2-like phenotype. This defect in macrophage phenotype and the proper transition results in delayed or even failure of wound healing. Specifically in the diabetic foot ulcer (DFUs), this macrophage dysfunction results in chronic infection and potentially amputation. The abnormal macrophage phenotype in diabetes is not fully understood but is believed to mainly result from epigenetic changes in macrophages and altered interactions between macrophages and other cell types, such as fibroblasts, endothelial cells, neutrophils and T-cells. Recent research on DFUs has focused on developing strategies to improve diabetic wound repair through modulation of macrophage polarization. Treatment of DFUs will greatly benefit from a multi-modal therapy that includes controlling high blood glucose, topical support, prevention of secondary infection, resolution of sustained inflammation and application of cellular therapies targeting macrophages.

**Keywords:** diabetic foot ulcer, diabetic wound healing, macrophage polarization, epigenetics, inflammation


**Author details**

Leandro Temer Jamas\*, Rodrigo Rhoden Barcellos, Carlos Roberto Padovani, Cassiano Victória and Helio Langoni Faculty of Veterinary Medicine and Animal Science (FMVZ), UNESP-Botucatu-Sp, Brasil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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.

*Serological Monitoring for* Leptospira *Spp. and Monitoring of Productive and Reproductive… DOI: http://dx.doi.org/10.5772/intechopen.98983*


*10.1.2 Synthetic anode materials*

It is quite evident that 3-dimensional carbon fiber (non-woven) can achieve a maximal current density of up to 31 A/m2 which is prepared by electrospinning and blowing the solution. The performance and efficiency of MFCs also depends on the system architecture, based on these 3D materials [78]. Double-sided air cathode reduces the boundaries of mass transfer. The stainless steel frame was

used for this design as a current assimilator and a carbon fiber support in the 3D matrix. In another study [79], it has been shown that an upgraded adaptation of the carbon-based multi-brush anode achieved admirable power generation. The power generated is similar to that obtained with a carbon anode with a single brush design. Because of cathodic limitations, the MFC system [80] gave a comprehensive comparison of carbon-based material for anodes, like graphite, carbon fiber veil, polycrystalline carbon rod, glossy carbon rod, graphite foil. The maximal current density attainable was calculated using a standardized biofilm grown in domestic wastewater. At 30°C, graphite, and polycrystalline carbon-based rods, both reached catalytic currents peaks of around 501 μA cm−2. By comparison, carbon fiber veil or paper-based material delivered a 40.1% higher current than graphite anode due to its large, microbial rich surface area [80]. In comparison with steady-state reactor, the rotational motion of carbon brush anodes in the tubular microbial fuel cell resulted in a 2.6 times rise in performance. The rotation was adequately mixing the nutrient and minimizing the limitation of mass transport. In general, several studies have shown that the existence and electrode content affected the kinetics of the biocatalyst. It has also been shown that the internal resistance is a major aspect affecting the overall performance. The use of 3-dimensional anode models, like carbon nanotubes (CNTs), nanofibers (CNF), gold/poly (e-caprolactone) microfibers (GPM), and gold/poly (e-caprolactone), to reduce the internal resistance increasingly preferred in microbial fuel cells. 3-dimensional anode material has less internal resistance than two-dimensional anodes. Such anode materials serve to increase the efficiency of nutrients, H+ , and O2 transfer via biofilm as compared to macroscopic carbon-based paper and planar gold-based anodes. Chemical assisted surface alteration of the CNT/CNF-based anodes has been demonstrated to reduce kinetic losses and cellular toxicity. Ren et al. [81] investigated vertically aligned CNT, randomly aligned CNT, and spin-spray layered CNT. The studied nanotube-materials have a 4000 m−1 very large surface area to volume ratio which is very huge. The results showed that CNT-based anodes attracted more electrogenic microbes than bare gold, resulting in a thicker and more stable formation of biofilms. Using CNTs in a miniature MFC device, a maximal power density of 3321 W/m3 was achieved [81]. This was 8.5 times greater than that attained with the 2D-electrode systems.

#### **10.2 Composite anodes**

Composite anodes have intrigued extensive interest recently. These materials were utilized to attain synergistic effects with two or more materials to alter original content, resulting in increased anodic kinetics efficiency.

#### *10.2.1 Graphite-polymer composites*

Tang, Yuan, Liu, & Zhou prepared a nano-structured capacitive layer of modified 3D anode consisting of core-shell nanoparticles derived from titanium dioxide (TiO2) and egg albumin (EWP). This was built into a loofah sponge carbon (LSC) to achieve an efficient 3-dimensional electrode. The LSC's coating with TiO2 and heat treatment caused tiny particles to cover its entire surface. The resulting altered anode supplied greater power than a graphite anode. The increased power was associated with the increased electrochemical capacity of 3-dimensional anodes and to the synergistic effects of carbon derived TiO2 and EMP with good characteristics like more surface area, improved biocompatibility, and favorable surface functionality for easier extracellular electron transport [82]. The anodes of opencelled carbon scaffold (CS) and carbon scaffold graphite (CS – GR) were created by

**111**

*Treatment of Dairy Wastewaters: Evaluating Microbial Fuel Cell Tools and Mechanism*

fibers achieved a maximal current density at a peak of 35.8 A/m2

whereas the power density improved by 1.49 times.

*10.2.3 Multi-walled carbon nanotubes composite*

*10.2.2 Carbon nanotubes composite*

significant attention lately.

*10.2.4 Graphene anodes*

as large surface area (up to 2600 m2

carbonizing the microcellular polyacrylonitrile (PAN) and composite PAN/graphite (PAN – GR). The PAN-GR was created by utilizing supercritical carbon dioxide (Sc-CO2), as a practical foaming agent. The maximal current density achieved with a CS altered anode was 102% greater than that with carbon felt. Improved performance has been referred to as enhanced hydrophilicity and biocompatibility caused by carbonization. Carbon nanofibers with improved graphite fibers and reduced nanotube-coated graphene oxide/carbon scaffold promise new composite anode materials. Using carbon nanofibers as anodes for MFC modified graphite

coated scaffold anode device with reduced graphene oxide/carbon obtained a power

used in combination with granular graphite (MFC-GG) in a tubular setup to boost the power density 5.2 and 1.3 times greater than that obtained with MFC-GG and MFC-GFB. The improved efficiency of the system was referred to the thick biofilm of the system, and scant internal resistance [83]. Six types of micro or nano-structured anodes utilized in micro-sized MFCs have been compared. The anodes under consideration included carbon nanotubes (CNTs), carbon nanofibers (CNFs), gold or poly (e-caprolactone) microfibers (GPM), nanofibers (GPN), planar gold (PG), and traditional carbon paper (CP). All anode's effectiveness was tested with the use of small and micro-liter sized MFC. A homemade 3-dimensional anode coating has been developed using the iron net as the structural anchor and fastened to a carbon felt layer [82]. The combination of carbon powder and a solution mixture of 30% polytetrafluoroethylene (PTFE) have greatly affected power generation. The performance was assessed using an acetate-fed MFC and the anode coating which improved the power generation considerably. The internal resistance measured in the MFC system was decreased by 59.3% compared to the non-coated iron net,

Due to their special intrinsic properties, including high conductivity, rust tolerance, high surface area and electrochemical inertness, the usage of CNTs has drawn

Multi-walled carbon nanotubes (MWCNTs) with carboxyl functional groups were utilized for MFC air respiration. It demonstrated a 2-fold improvement in power density relative to the carbon cloth electrode [84]. In a recent report, multiwalled carbon nanotubes/SnO2 nanocomposite coated on the glass fiber electrode

respectively [86]. The use of graphite coated with manganese oxide/multiwalled carbon nanotubes composites has greatly elevated benthic microbial fuel cells in another study. The composite provided greater hydrophobicity, kinetic movement, and power density when opposed to the standard graphite electrode. The shift seen was attributed to the consolidated impact of the Mn ions electron transfer shuttle

Graphene is an allotrope of 2D crystalline carbon with unusual characteristics such

(7200 S/m), and exceptional tensile strength up to 35 GPa [88]. Graphene-modified

/g), exceptionally high electrical conductivity

is used [85] producing maximal power densities of 1422 mW/m2

on the reaction site and its redox reactions (i.e. anode and biofilm) [87].

. Composite graphite fiber brush anode (MFC-GFB) was

. The nanotube-

and 457 mW/m2

,

*DOI: http://dx.doi.org/10.5772/intechopen.93911*

density of 335 mW/m3

#### *Treatment of Dairy Wastewaters: Evaluating Microbial Fuel Cell Tools and Mechanism DOI: http://dx.doi.org/10.5772/intechopen.93911*

carbonizing the microcellular polyacrylonitrile (PAN) and composite PAN/graphite (PAN – GR). The PAN-GR was created by utilizing supercritical carbon dioxide (Sc-CO2), as a practical foaming agent. The maximal current density achieved with a CS altered anode was 102% greater than that with carbon felt. Improved performance has been referred to as enhanced hydrophilicity and biocompatibility caused by carbonization. Carbon nanofibers with improved graphite fibers and reduced nanotube-coated graphene oxide/carbon scaffold promise new composite anode materials. Using carbon nanofibers as anodes for MFC modified graphite fibers achieved a maximal current density at a peak of 35.8 A/m2 . The nanotubecoated scaffold anode device with reduced graphene oxide/carbon obtained a power density of 335 mW/m3 . Composite graphite fiber brush anode (MFC-GFB) was used in combination with granular graphite (MFC-GG) in a tubular setup to boost the power density 5.2 and 1.3 times greater than that obtained with MFC-GG and MFC-GFB. The improved efficiency of the system was referred to the thick biofilm of the system, and scant internal resistance [83]. Six types of micro or nano-structured anodes utilized in micro-sized MFCs have been compared. The anodes under consideration included carbon nanotubes (CNTs), carbon nanofibers (CNFs), gold or poly (e-caprolactone) microfibers (GPM), nanofibers (GPN), planar gold (PG), and traditional carbon paper (CP). All anode's effectiveness was tested with the use of small and micro-liter sized MFC. A homemade 3-dimensional anode coating has been developed using the iron net as the structural anchor and fastened to a carbon felt layer [82]. The combination of carbon powder and a solution mixture of 30% polytetrafluoroethylene (PTFE) have greatly affected power generation. The performance was assessed using an acetate-fed MFC and the anode coating which improved the power generation considerably. The internal resistance measured in the MFC system was decreased by 59.3% compared to the non-coated iron net, whereas the power density improved by 1.49 times.


*3.11. Statistical Analyses*

Statistical analyses were performed in triplicate for each sample, and the results are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to determine differences between the mean values of triplicate groups. Statistical significance was established at *P* < 0.05 with the least significant difference (LSD) procedure of SPSS version 19.0.


**5.2 Nanoparticle surface**

Nanoparticles have a very large surface area compared to the size of the nanoparticle and compared to normal particles, and this space provides a high degree of interaction with the molecule and its environment, and of course it is possible to almost determine the final fate of the nanoparticle inside the body through determining the strength of the interaction between the nanoparticle and its surroundings, and it depends largely on a mixture of size. And surface properties.  This provides a tremendous driving force for diffusion, especially at elevated temperatures [31, 32].

**169**

*The Roles of Nanoparticle in the Treatment and Diagnosis of Ovarian Cancer*

The rare properties of nanoparticles have been exploited to present the science of chemical therapeutics in a unique way and as we previously knew that nanoparticles and the reason for their special composition can exploit the vascular infusion and absorption mechanisms associated with tumor cells to enter and implement a specific therapeutic effect where the particles accumulate in the tumor tissue, taking advantage of the enhanced permeability and retention effect, of course, when comparing the usual systemic chemotherapy by systemic administration with the science of chemotherapy coated with nanoparticles, where it can deliver the desired dose to the tissue environment of the tumor. In almost the same way, special bonds to cancer cells are added to nanoparticles to arrive in a uniform and targeted way to reduce the toxicity of systemic chemotherapy, we will mention the most common

*The idea of special targeting of cancer cells to the nanoparticles that can be filtered through the kidney. As the*

*nanoparticles target cancerous diseases, the residue that is not targeted is removed.*

A present example is a (PEGylated liposomal doxorubicin) formulation that

U.S. Food and Drug Administration (FDA) approved for use in recurrent and

The liposomes coated with doxoribicin remain in circulation for 2–3 weeks after the injection process until the end of their estimated effective life, these particles enter the tumor tissue and settle in it through defects and gaps in the tumor vessels and then settle near the blood vessel, The extravasated liposomes release the drug

We previously knew that the liposomes remain less time in the circulatory system, and this affects the drug levels that reach the tumor tissue, and this can be bypassed by reducing the size of the carrier, but this may affect the levels of the drug and its required quantity, so to get rid of problems, the carrier is coated with polymers such as polyethylene glycol (PEG). As this system works to mask the immune system and increase circulation time [35]. The proposed mechanism of

*DOI: http://dx.doi.org/10.5772/intechopen.97117*

**5.3 Nano-chemical therapeutics**

platinum-resistant cancers (**Figure 4**) [34].

action and accumulation of DOXIL is as follows:

therapeutics [33].

has been.

**Figure 3.**

*The Roles of Nanoparticle in the Treatment and Diagnosis of Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.97117*

### **Figure 3.**

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

minimum is 10 nanometers as a threshold for renal excretion and where it is known that vessels in tumor cases are subject to leak Macromolecules in a certain way, so the nanoparticles should not be able to circulate for a long time in the bloodstream and have a chance to reach the bloodstream through the vessels of the tumor tissue and enter the tumor tissue where the size of the nanoparticles is greater than 6–12 nm, which is the diameter of the sieve in the blood vessels of normal tissues and It is prevented from entering and not damaging the normal tissue, and it is known that the diameter of the sieve in the tumor blood vessels ranges from 40 to

*Basic nanoparticles used in clinical trials. (a) Nanoparticles composed of therapeutic components. (b) the nanoparticles that are formed from polymer/drug.

Nanoparticles have a very large surface area compared to the size of the nanoparticle and compared to normal particles, and this space provides a high degree of interaction with the molecule and its environment, and of course it is possible to almost determine the final fate of the nanoparticle inside the body through determining the strength of the interaction between the nanoparticle and its surroundings, and it depends largely on a mixture of size. And surface properties.  This provides a tremendous driving force for diffu-

**168**

200 nm (**Figure 3**) [30].

**Figure 2.**

**5.2 Nanoparticle surface**

sion, especially at elevated temperatures [31, 32].

*The idea of special targeting of cancer cells to the nanoparticles that can be filtered through the kidney. As the nanoparticles target cancerous diseases, the residue that is not targeted is removed.*

### **5.3 Nano-chemical therapeutics**

The rare properties of nanoparticles have been exploited to present the science of chemical therapeutics in a unique way and as we previously knew that nanoparticles and the reason for their special composition can exploit the vascular infusion and absorption mechanisms associated with tumor cells to enter and implement a specific therapeutic effect where the particles accumulate in the tumor tissue, taking advantage of the enhanced permeability and retention effect, of course, when comparing the usual systemic chemotherapy by systemic administration with the science of chemotherapy coated with nanoparticles, where it can deliver the desired dose to the tissue environment of the tumor. In almost the same way, special bonds to cancer cells are added to nanoparticles to arrive in a uniform and targeted way to reduce the toxicity of systemic chemotherapy, we will mention the most common therapeutics [33].

A present example is a (PEGylated liposomal doxorubicin) formulation that has been.

U.S. Food and Drug Administration (FDA) approved for use in recurrent and platinum-resistant cancers (**Figure 4**) [34].

We previously knew that the liposomes remain less time in the circulatory system, and this affects the drug levels that reach the tumor tissue, and this can be bypassed by reducing the size of the carrier, but this may affect the levels of the drug and its required quantity, so to get rid of problems, the carrier is coated with polymers such as polyethylene glycol (PEG). As this system works to mask the immune system and increase circulation time [35]. The proposed mechanism of action and accumulation of DOXIL is as follows:

The liposomes coated with doxoribicin remain in circulation for 2–3 weeks after the injection process until the end of their estimated effective life, these particles enter the tumor tissue and settle in it through defects and gaps in the tumor vessels and then settle near the blood vessel, The extravasated liposomes release the drug

**Figure 4.** *This shape represents a Doxil liposome where doxorubicin is confined and encapsulated in the internal compartment where drug molecules are tightly packed.*

components, and drug molecules enter deeply into the tumor tissue, where they reach and kill cancer cells. It is noted that this mechanism does not need a physical encounter and contact between the liposome and the cell, where the drug can reach and penetrate the barriers that intercept the particles [36].

There is another example of a nano-drug transporter (micelle) these structures typically contain a more hydrophobic component that helps solubilize/encapsulate therapeutic compounds, while a hydrophilic component provides stability of the assembly in aqueous environments and offers conjugation sites for eventual targeting ligands. This type of nanostructure has been widely used recently, an example being the D-α-tocopheryl polyethylene glycol (PEG) 1000 succinate (TPGS) (**Figure 5**) [37].

It is an amphiphilic water-soluble derivative of natural source vitamin E and PEG, that has been widely employed as a micelle-former biomaterial. Also, it has been reported that TPGS can inhibit the efflux pump that mediates multidrug

*A typical structure of polymeric micelle representing the drug encapsulated and targeting moiety attached.*

**171**

**Author details**

Mohammed E. Mansur University of Kufa, Najaf, Iraq

provided the original work is properly cited.

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 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,

*The Roles of Nanoparticle in the Treatment and Diagnosis of Ovarian Cancer*

tissue and high efficiency to stay in vivo for acceptable periods.

resistance in tumor cells, known as P-glycoprotein (P-gp), In this context, TPGS has been employed for DOX encapsulation within polymeric micelles, Single polymers are the most acceptable type in recent times because they increase the solubility and stability of hydrophobic drugs, increase cellular absorption capacity, and to increase the susceptibility, two micelles were combined to obtain mixed micelles to increase strength. as enhanced thermodynamic and kinetic stabilities, higher drug loading (DL) capacity, more accurate size control and easier ways to modify their surface

From the foregoing that nanoparticles are more efficient in the diagnosis and treatment of ovarian cancer as a basic alternative to chemotherapy and a highly efficient pre and postoperative adjuvant due to their great ability to reach the target

*DOI: http://dx.doi.org/10.5772/intechopen.97117*

with different moieties [38, 39].

**6. Conclusion**

*The Roles of Nanoparticle in the Treatment and Diagnosis of Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.97117*

resistance in tumor cells, known as P-glycoprotein (P-gp), In this context, TPGS has been employed for DOX encapsulation within polymeric micelles, Single polymers are the most acceptable type in recent times because they increase the solubility and stability of hydrophobic drugs, increase cellular absorption capacity, and to increase the susceptibility, two micelles were combined to obtain mixed micelles to increase strength. as enhanced thermodynamic and kinetic stabilities, higher drug loading (DL) capacity, more accurate size control and easier ways to modify their surface with different moieties [38, 39].


**3. Mathematical modeling**

The mathematical model analyzed is based on the atomic force microscopy system considering a viscoelastic term that is assigned to the medium in which the tip performs the analysis. For this, the proposed model considers the interatomic forces between the probe and the surface of the samples. These interatomic forces are of the van der Waals type that arises from the Lennard-Jones potential. Eqs. (1) and (2) describe the Lennard Jones potential (ULJ) and the van der Waals force (*FWD*), respectively [20, 21].

$$U\_{Lf} = \frac{A\_1 R}{1260z^7} - \frac{A\_2 R}{1260z} \tag{1}$$

$$F\_{WD} = \frac{-\partial U\_{Lf}}{\partial \mathbf{z}} = \frac{A\_1 \mathbf{R}}{180 \mathbf{z}^8} - \frac{A\_2 \mathbf{R}}{6 \mathbf{z}^2} \tag{2}$$

Where *R* is the radius of the tip, *z* is the distance of the tip, *A*1 ¼ *π*2*ρ*1*ρ*2*c*1, and *A*2 ¼ *π*2*ρ*1*ρ*2*c*2 are called Hamarke constants (where *ρ*1 and *ρ*2 are the number densities of the two interacting kinds of particles, and *c1* and *c2* are the London coefficient). Therefore, the mathematical model analyzed is the one considered by Ref. [20] in which the deflection is determined by the following:

$$w(\mathbf{x}, t) = u(\mathbf{x}, t) + w(\mathbf{x}) + y(t) \tag{3}$$

where *w x*ð Þ , *t* is microcantilever beam deflection, *u x*ð Þ , *t* is a relative deflection of displacement of the actuator, described as *y t*ðÞ¼ *Y* sin ð Þ *Ωt* . We consider the term viscoelastic to the AFM system considering *z* [15, 17]:

$$F\_{\rm CS} = \frac{-\mu\_{\rm eff} b^3 L}{\left[x - w(L) - u(L, \ t) - Y \sin(\Omega t)\right]^3} \dot{u} \tag{4}$$

Where *μeff* is the coefficient of effective viscosity, and *b* and *L* are the width and length of microcantilever, respectively. Considering the vibrations on the intermittent configuration, described by:

$$
\rho A \ddot{u}(\mathbf{x}, t) + EI(u^{\prime\prime\prime}(\mathbf{x}, t) + w^{\prime\prime\prime}(\mathbf{x})) = \left(\frac{-A\_1 R}{180(Z - w(L) - u(L, \ t) - Y \sin\left(\Omega t\right))^3}\right)^2
$$

$$
+ \frac{A\_2 R}{6(Z - w(L) - u(L, \ t) - Y \sin\left(\Omega t\right))^2}
$$

$$
$$

$$
+ \rho A \Omega^2 Y \dot{u}(\mathbf{\Omega t})\tag{5}
$$

Eq. (5) is nonlinear and nonautonomous, and its discretization can be achieved through a dynamic projection on the linear modes of the system. According to Ref. [19], an approximation of the solution of Eq. (5) is by using the linear modes and frequencies of the microcantilever around its electrostatic equilibrium that are different from those of a microcantilever located far from the surface. Therefore,

*MEMS-Based Atomic Force Microscope: Nonlinear Dynamics Analysis and Its Control DOI: http://dx.doi.org/10.5772/intechopen.108880*

calculations of linear modes and microcantilever frequencies over nonlinear electrostatic equilibrium are rigorously calculated using Galerkin's method. The Galerkin's method is used to analyze problems of beams subjected to moving loads with timevarying velocities.

However, we consider under near-resonant forcing, and in the absence of additional internal resonances, only one mode of the microcantilever is assumed to participate in the response.

$$U(\mathbf{x}, t) = \phi\_1(\mathbf{x}) q\_1(t) \tag{6}$$

where *ϕ*1ð Þ *x* is the first approximate eigenfunction about the chosen equilibrium. Substitution of (6) into (5), multiplication of (5) by *ϕ*1ð Þ *x* , subsequent integration over the domain, and the introduction of a modal damping consistent with the Q factors listed in **Table 1** yields the single-degree-of-freedom model:

$$\ddot{\bar{\eta}} = -d\_1 \dot{\bar{\eta}} - \bar{\eta} + B\_1 + \frac{C\_1}{\left(1 - \bar{\eta} - \bar{\eta}\sin\left(\Omega \tau\right)\right)^8} + \frac{C\_2}{\left(1 - \bar{\eta} - \bar{\eta}\sin\left(\Omega \tau\right)\right)^2} \tag{7}$$

$$-\frac{p\dot{\bar{\eta}}}{\left(1 - \bar{\eta} - \bar{\eta}\sin\left(\Omega \tau\right)\right)^3} + \bar{\eta}\bar{\Omega}^2 E\_1 \sin\left(\Omega \tau\right)$$

where : �*η* ¼ *x*1ð Þ*τ η* , *x*1 ¼ *ϕ*1ð Þ *L q*1ð Þ*τ* , *η* ¼ *z* � *wL*, *x*1 ¼ *ϕ*1ð Þ *L q*1ð Þ*τ* , *η* ¼ *z* � *w L*ð Þ, *τ* ¼ *ω*1*t*, *Ω*� ¼ *Ω ω*1 *d*1 ¼ *C*1 *ω*1*ρA* Ð *L* 0 *ϕ*2 1*dx*, *B*1 ¼ ð Þ 1 � *α*� *Γ*1, *C*1 ¼ *A*1*R* 180*k*ð Þ*η* 9 *Γ*1,*C*2 ¼ *A*2*R* 6*k*ð Þ*η* 3 *Γ*1, *ω*2 1 ¼ *EI*Ð *L* 0 *ϕ*1*ϕ*cr 1 *dx ρA* Ð *L* 0 *ϕ*2 1*dx* , *α*� ¼ *z η* , *Γ*1 ¼ *kϕ*2 1 ð Þ *L ω*2 1*ρA* Ð *L* 0 *ϕ*2 1*dx*, *k* ¼ 3*EI L*3 , *ζ* ¼ *Y η*, *E*1 ¼ *ϕ*1ð Þ *L* Ð *L* 0 *ϕ*1*dx* Ð *L* 0 *ϕ*2 1*dx* , and *p* ¼ *μeff b*2 *l*.

Thus, considering, �*η* � *x*1 and �*η*\_ � *x*2 we can rewrite Eq. (7) in the following system of differential equations:

#### **Table 1.**

*Parameters used for numerical analysis.*

**Figure 2.**

*Scheme of the AFM system. (a) System in initial start and (b) intermittent.*

$$\frac{d\mathbf{x}\_1}{dt} = \mathbf{x}\_2$$

$$\frac{d\mathbf{x}\_2}{dt} = -d\_1\mathbf{x}\_2 - \mathbf{x}\_1 + B\_1 + \frac{C\_1}{\left(1 - \mathbf{x}\_1 - \zeta \sin\left(\varDelta \mathbf{r}\right)\right)^8} + \frac{C\_2}{\left(1 - \mathbf{x}\_1 - \zeta \sin\left(\varDelta \mathbf{r}\right)\right)^2} \tag{8}$$

$$\frac{-p\mathbf{x}\_2}{\left(1 - \mathbf{x}\_1 - \zeta \sin\left(\varDelta \mathbf{r}\right)\right)^3} + \zeta \bar{\mathcal{Q}}^2 E\_1 \sin\left(\varDelta \mathbf{r}\right)$$

**Figure 2** shows the schemes simplify of deflection behavior of nano cantilever of AFM″ for completeness.


**1.6 Equipment records**

Equipment records consist the files including inventory, job performed, and maintenance cost. All these activities are well mannered which can be done while performing the jobs. Also, the maintenance costs are categorized in a historic profile manner. In general, stores and accounting departments provide the information regarding inventory. Operations, drawings, service manuals, warranties and so on are included in the files.

#### **1.7 Maintenance job planning**

possibilities of running out of stock caused from the dwell lead time and the random variability of demand. The outcome of the stock holding is as shown in **Figure 1**. Here, solid lines describe the stock held, and the dotted lines reveal about stock

*q* ¼ ffiffiffiffiffiffiffiffiffiffiffiffi

where q = reorder quantity; Co **=** ordering cost; D = mean demand per unit time;

*M* ¼ *DL* þ *Kσ* ffiffiffi

where L **=** lead time, *σ* ¼standard deviation of demand per unit time, k **=** normal

A work order is approved and given the directions in a separate or group vise to carry out the tasks. In this, all maintenance activities and responsibilities are covered to control the costs and to enhance the job performance. Work order should at least contain information such as requested and planned completion dates, work description and its reasons, planned start date, labor and material costs, item or items to be affected, work category, and appropriate approval signatures.

1.The documents required to facilitate work planning, execution and control,

2.The basic documents required for the work order system include the work order, materials and tools requisition forms, job card, maintenance schedule,

maintenance program, plant inventory, and equipment history files.

*L*

2*D*Co p *=*Ch (1)

p (2)

on-hand.

**Figure 1.**

Ch **=** holding cost.

standard variety.

**310**

The expression for re-order quantity is:

*Operations Management - Emerging Trend in the Digital Era*

*Re-order level (source MTB by Alembazezwu).*

Assume stack is re-ordered at regular time interval. Lead time between order and delivery is negligible.

Negative stack is not allowed by the policy.

Constant rate of demand per unit. The reordering level M is calculated as:

**Maintenance work order system**:

The two main parts of work order system are:

and the work order flow process.

Job planning is an essential element of the effective maintenance management. A number of tasks may have to be performed prior to commencement of a maintenance job; for example, procurement of parts, tools, and materials, coordination and delivery of parts, tools, and materials, identification of methods and sequencing, and coordination with other departments. Job planning is considered as a main technique to make good observations and preventive maintenance. Here, the primary job is to complete what parts to be maintained and what is the best method to enhance the performance of the jobs. Skills, talent, and good deal time are necessary to accomplish the better procedures. However, this should be performed to gain the good experience to train the future design engineers. But, writing skills are also an important qualification along with practical experience in many practices. The way of writing the language is short, concise, and clear to understand easily. All the statements should be written clearly and the following should be followed strictly:

When the procedure is completed and turned into maintenance control, the planner or scheduler should note any additional work required and see that it gets done according to priority.

#### **1.8 Maintenance scheduling**

Maintenance scheduling is as important as job planning. Schedule effectiveness is based on the reliability of the planning function. Fixed interval maintenance tasks and schedules are generally to be considered only when the option has to control the failures detected in advance. Hence, the planning for identifications and preventive levels can be finalized in days, weeks, and even months together soon to make sure that the time for production must be convenient.


*4.1. Spatial Autocorrelation Test and Spatial Econometric Model Selection*

Urban carbon emission intensity and green economic efficiency are spatially correlated, which is a prerequisite for spatial econometric modeling [60]. Stata.23 is used to calculate the global Moran index to explore the spatial agglomeration characteristics. As shown in Table 2, the GEE Moran index is positive for all the years from 2007 to 2019, and the spatial agglomeration strengthens year by year except for 2013–2015, all of which pass the 1% significance level test. During the observation period, the Moran index of Carin is positive in all cases, and the degree of spatial agglomeration increases in fluctuation. Overall, both urban green economic efficiency and carbon emission intensity have significant spatial agglomeration characteristics.

**Table 2.** Global Moran Index test results for green economy efficiency and carbon emission intensity.

Note: \*\*\*\*, \*\*, and \* represent significance levels of 1%, 5%, and 10%, respectively.

Based on passing Moran's I test, the spatial effect econometric model can be selected. Additionally, to measure the nonlinear characteristics of tourism development on urban carbon emission intensity and green economic efficiency, models (3) and (4) containing quadratic terms of tourism development variables were introduced. First, the LM test, LR

test and Wald test were performed to identify the spatial econometric models. Both the LM spatial lag test and LM spatial error test showed high significance, so both the SAR model and SEM were suitable for this study, and we chose the SDM model that combined both. Then, the LR test and Wald test were applied to further determine whether the SDM could be degraded to the SAR model or SEM. The comprehensive test results showed that the SDM was better than the SAR and SEM, so the SDM was selected as the baseline regression model in this paper. Based on the SDM model [62], all matrices passed the Hausman test at a 1% significance level except for model (1), which had a negative value, so the fixed-effects model was selected. As shown in Table 3, the overall R2 of the individual fixed-effects model is significantly better than that of the time-point fixed-effects and double fixed-effects models. Finally, the individual fixed-effects spatial Durbin model is chosen to analyze the impact of tourism development on urban carbon emission intensity and green economic efficiency.

**Table 3.** Spatial econometric model selection.

Note: \*\*\*\*, \*\*, and \* represent significance levels of 1%, 5%, and 10%, respectively.


**2.3. Stillage from corn-to-ethanol process**

remove up to 90% of biochemical oxygen demand (BOD) but the chemical oxygen demand (COD) removal efficiency is just in the range of 20–50% [16–19]. Secondly, the United States Environmental Protection Agency (US EPA) have strict and specific policies about these industrial wastes, such as the US EPA CMOM (capacity, management, operation, and maintenance program, including grease control program), the US EPA final pulp and paper cluster rule and amendments, the US EPA CWA (Clean Water Act), the FOG ordinance/FOG management policy, and so on. That could be considered as the driving force to push industries to treat these wastes before discarding. Finally, these materials from industrial waste contain high organic content, which means they have the potential to be treated anaerobically as the energy feedstock.

Pulp and paper industry produces a large quantity of wastewater of high organic strength

every ton of paper produced [22]. In the paper manufacturing processes, pulping and bleaching processes creates most of the wastewater streams [23, 24]. These wastewaters typically have high organic content (COD 800–4400 mg−1) [24–26], high biological content (BOD 300– 2800 mg−1) [24–26], and high dye content (1200–6500 color unit) [24–26]. Several steps of treatment process were generally involved, including a primary clarification process to remove the suspended solids, a secondary treatment process to remove most of the air lagoons, and a final biological treatment process (aerobic) to remove the biological content (BOD5) [25, 26]. However, due to the recalcitrant chemical properties, the final effluent always still contains

Anaerobic treatment technique has not been widely used in the pulp and paper industry yet [27, 28]. One major advantage of anaerobic treatment is that the process is capable of treating high-organic strength streams that are not suitable for aerobic processes [30, 31]. Furthermore, it has the added benefit of lower treatment cost because the produced biogas can be diverted to energy generation [32]. Traditional treatment technique of energy-rich wastes should be avoided as far as possible mainly because of their low energy recovery efficiency [33], but the recovered biogas from anaerobic digestion process has a high methane content (60–80%) and can be directly used as fuel [34]. One of the current research issues is most of the evaluations for pulp and paper wastewater are only focused on synthetic waste stream in the lab-scale environment (reactor size 5–50 L) [29, 35–40], which makes the results less representative to large-scale industrial fabrications, a research utilizing pilot-scale system, and practical waste

Using biogas as an alternative source of energy is gaining more attention globally in recent decades [41, 42]. There have been an increasing number of studies performed to evaluate the

of wastewater is generated for

[20, 21]. Even with the most modern operations, about 60 m3

large amount of high molecular weight organic compounds [25].

streams directly from the paper mill would be more helpful and relevant.

**2. Substrates**

**2.2. Brown grease**

**2.1. Paper mill effluents**

254 Advances in Biofuels and Bioenergy

Based on the increased demand of renewable energy, bio-ethanol as an alternative energy source was considered and has enormous economic and strategic advantages. In the past decade, the national total annual fuel-grade ethanol production has increased from 1.77 billion gallons (in 2001) to 13.95 billion gallons (in 2011). In 2005, 67% of this ethanol was produced from dry mill corn [63], and this percentage has kept on increasing because of the low cost of this technology [64].

In a typical bio-ethanol production process, corn mash has been fermented and distilled to produce high purity ethanol, and the fermentation residue is called whole stillage, which is centrifuged to produce wet cake (precipitate) and thin stillage (supernatant). About 50% of the thin stillage is recycled as backset. The remainder is further concentrated by evaporation to produce syrup and blended with dried wet cake to create a feed product known as distiller's dry grain with soluble (DDGS). The effluent of evaporation process was purified and recycled as water reuse.

Stillage handling is the most energy consuming process in the life cycle of corn to ethanol process. The drying and evaporation of stillage will take more than 35% of the total energy consumption [65], which makes the stillage treatment technique a main limitation of bioethanol making process [66]. Except for energy consumption, thin stillage is also a kind of high strength wastewater, which exhibits a considerable pollution potential [67]. Up to 20 l of stillage will be produced for each l of corn ethanol [68, 69], and the pollution potential of generated stillage can reach to a chemical oxygen demand (COD) of over 100 g L−1 [69].

as an upflow flooded-bed reactor, the HRT is about 1.7–2.4 days. The feeding and operational

Biogas Recovery from Anaerobic Digestion of Selected Industrial Wastes

http://dx.doi.org/10.5772/intechopen.72292

257

The evaluation lasted for 156 days, and was divided into six periods according to different feeds and operating conditions. Initially, the packed-bed column was operated as a downflow digester, with a recirculation ratio of 5.0. Note for this stage, there is no water retention. Beginning with the 80th day, the AD column was operated upflow direction; the HRT was kept at 1.7–2.4 days. The entire operation is built on neutral pH range (6.92–7.60, see **Table 1**) and slightly mesophilic condition (T = 31.5–34.5°C, measured for effluent, see **Table 1**).

**Table 2** listed the initial characteristics of each kind of substrates. The COD concentrations for each type of substrate ranged from 2800 to 4500 mg L−1. In this study, the waste streams from paper mill are mostly in liquid phase and have relatively very low solid content (TS < 1 wt%, see **Table 2**). As mentioned above, a sugar water substrate was used to adjust the pH of the mixed substrate. The sugar water (SW) is a high organic content and slightly acidic substrate (COD = 408,000 mg L−1, pH = 3.99). In this study, the sugar water was blended for about 0.5 wt%.

digested (mass basis) against the time axis. Note the system start-up and recovery during substrate changes were not included in the figure. There are totally six linear stages (Stage I–Stage

Based on the treatability study listed above, all waste streams are readily treatable. The anaerobic treatment removed 50–65% of substrate COD. Coupled with the aerobic treatment using a CSTR ASP, the overall COD removal efficiency was 55–70%. The application of anaerobic treatment has the potential of significantly improving the energy footprints of the pulp and paper industry.

Substrate FC + SW EOP + SW EOP + SW EOP + DO + SW EOP + DO EOP + DO + SPL

d) 2.96 ± 0.701 3.02 ± 0.381 2.25 ± 0.812 2.75 ± 0.702 1.59 ± 0.482 1.44 ± 0.482

32.7 ± 2.6 34.3 ± 1.6 31.5 ± 3.1 34.5 ± 1.6 32.2 ± 1.8 33.3 ± 0.9

Flow scheme Downflow Downflow Upflow Upflow Upflow Upflow

HRT (d) — — 2.44 ± 0.83 1.72 ± 0.51 2.12 ± 0.91 1.82 ± 0.55 pH in digester 6.92 ± 0.39 7.23 ± 0.11 7.42 ± 0.10 7.60 ± 0.48 7.25 ± 0.02 7.26 ± 0.09

production and the cumulative COD

yield was calculated as the ratio of

production rate; these six

kg-COD-1

characteristics are summarized in **Table 1**.

**Figure 1** shows the plots between cumulative CH4

periods were considered as steady state periods. The CH4

for the substrates evaluated.

OLR (kg-COD/m3

(°C)

1

2

Temperature of effluent

Based on the volume of packing media.

Based on total volume of the packed-bed digester. Note: Day 37–44 was in maintenance and recovery mode.

**Table 1.** Pilot-scale feeding activities and conditions during six operating periods.

VI, see **Figure 1**) that the system has a stable and consistent CH4

the slopes of the two curves in **Figure 1**. The values range from 0.22 to 0.34 m3

**Operating periods 1 2 3 4 5 6** Duration (days) 1–36 45–81 82–135 136–142 143–148 149–156

The problems described in the previous paragraph have a significant negative impact on the industrial cost of the corn to ethanol process. Thus, a gate-to-gate life cycle assessment for thin stillage treatment was needed to provide a synergistic effect for energy recovery and cost saving. AD technique could be used to remove COD from thin stillage and also to convert the organic fraction of thin stillage into methane, which is a readily in-plant-usable energy source for ethanol industries [69]. Once this AD process was linked as a gate-to-gate life cycle to the ethanol production chain, the efficiency of the complete cradle-to-gate evaluation will be improved and the total cost will be reduced.


**Preface XI**

	- **Part 2 Lithographies for Biological Applications 15**
	- **Part 3 Self-Assembled Colloidal Substrates for Nanofabrication 51**
	- **Part 4 Periodic Structures for Diffractive Optics and Surface Wetting 117**

Chapter 8 **Photocontrolled Reversible Dimensional Changes of Microstructured Photochromic Polymers 149**  Despina Fragouli, Roberto Cingolani and Athanassia Athanassiou

### **Part 5 Extreme UV Lithography 165**

Chapter 9 **Approach to EUV Lithography Simulation 167**  Atsushi Sekiguchi


**2. Materials and Methods**

#### *2.1. Study Area*

The Brenne portion of Claise basin (Figure 1) covers an area of 707 km2 and is one of the three entities housing the 4500 ponds of the Brenne Natural Regional Park, which are mostly grouped in the form of interconnected chains [36]. Within the Claise, 2179 of the Brenne waterbodies, along with the Five Bonds channel (Blizon), are the main contributors [49] to the 87.6 km long Claise River having an average discharge of 4.5 m3/s [50]. The basin is mostly dominated by a degraded oceanic climate with a mean temperature of 11 ◦C and average annual precipitation of 700 mm [51]. As to its topographical profile, the basin is considered to be a flat plain, with 99% of its surface falling into the 0%–5% slope class, while its altitude varies between 76 m and 181 m. The six poorly-permeable soil classes of the basin are mostly dominated by Luvisols [36,52]. At a combined state, the Claise's challenging pedology, flat topographical setting, and quasi–impermeable lithology have resulted in the stagnation of incoming water leading to the formation of natural ponds [53]. According to Bennarrous (2009) [37], however, these ponds are not only a product of natural processes but also an anthropogenic adaptation to a poorly drained domain and a source of economic livelihood (aquaculture) in an environment of limited productivity. As a result of intensive pond proliferation throughout time, the basin has acquired a particular hydrographic network characterized by an abundance of different kinds of water bodies. Despite its richness, nonetheless, the hydrographic network of the basin is randomly organized and presents severe fragmentation [54]. In contrast to the evolving pond proliferation, the land occupation setting of the Claise has been relatively unchanged for the last 19 years, mainly displaying a dominance of an interlocked mosaic of forests, grasslands, and agricultural areas [54].


**4. Research results**

There are two themes that emerged during data analysis from an argument of findings for this investigation [22]. In consideration of reading proficiency levels, it emerged that reading is affected by disbanded learner groupings due to the COVID-19 pandemic era code of practice. The second finding outlined relates to learner attitudes resulting in feeling isolated from their peers, and yet this is proving to somehow influence language development intertwined with learner achievement.

#### **4.1 Proficiency levels in reading**

Among the group of participants, one teacher reported own observance on proficiency levels in reading among learners since in the classroom setting, learner groupings had no longer become the talk of the schooling environment: *My greatest worry is that learners in my class previously performed maximally well during the times when I used to group them as per their reading abilities.* On the same view, another

*Social Distancing Disbanding Learner Groupings: A Case on Language Development DOI: http://dx.doi.org/10.5772/intechopen.104893*

participant concurred: *With time, all those who still displayed some difficulties in reading texts would improve through interactions and support made by members in a single group. Learners would later require me as their teacher to allow time to engage in competing as groups as a way to encourage learners to be all in an accepted level with regards to their proficiencies in comprehending with texts.*

#### **4.2 Learner attitudes versus isolation**

It was divulged that the majority of learners felt isolated from their peers. Henceforth, the current COVID-19 social distancing restrictions pertaining to the seating logic of one learner seated in a stipulated distance away from the other, they no longer had opportunities to either exchange ideas or share information as they used to when seated as groups.

In relation to this encounter, one participant argued that: *Even if those learners who are super gifted than the rest have successfully comprehended with the text within a short space of time, as their teacher I have recently developed reluctance to either pair or group learners according to their reading abilities, with fear of clomping learners together, in case one contaminates the deadly disease from the other.* On that note one participant reported a story that left us so sad, but without any remedy to suggest: *As I came a little bit closer, one by one, to those learners with difficulties to comprehend with the assigned texts, they almost gave me a similar response that they felt so isolated and deserted as groupmates who used to assist them were now seated afar, making it difficult to easily exchange ideas and ask for assistance and mentorship when they came across new, unfamiliar and difficult words.*

#### **5. Discussion**

Pertaining to reading proficiency levels, a key finding was a noted deterioration in reading progress among learners. Teachers offering English language as a subject debated that motivation within learners as groups no longer exists, actually, it has then been compromised as each learner had to battle or struggle on comprehending the text all alone, this somehow displaying a sudden lack of collegiality among learners registered for the same subject. This situation was even made worse by the current situation that existed when learners were at their cultural or rather home backgrounds. Learners had reported to respective teachers that isolation also roams in their living environments to such an extent that even if one had a neighbor with whom they are enrolled for the same grade, phase, or subject, parents denied their children access to visit others for purposes of information sharing sessions [23]. As authors of this manuscript, we really feel pity for the cohort of learners experiencing difficulties in comprehending with texts. Actually, this then becomes a call for language teachers to identify alternative strategies that would curb the situation of learners who feel isolated and left alone, as this might lead to implications of increased learner-dropout rate as well as lowered progression levels. On the other side, a reading nation is a wealthy nation, we all need to engage with written texts in order to be an economically balanced country.

To curb the situation, learners need to be motivated so as to be able to adapt in the unfamiliar systems of self-independency. Motivation is known for its importance especially in learning an unfamiliar language, in this instance this being an English language. Motivated learners have since been noted of achieving their goals, while on the other hand those with lack of motivation become victims of not obtaining the intended academic achievement. Nurtured motivation has been identified to lead higher levels of language competence. It therefore lies with English

language teachers that as learners are held in situations of no longer vibrantly engaging with group members in the classroom environment, learners be encouraged and driven toward attaining short-term goals such as reading and writing, by so doing, they would improve in language proficiencies. Dissolution of learner groupings is perceived by learners as not supportive toward language learning. With limited collaboration, some learners could undergo stress disorders or related sicknesses [6–10].

Furthermore, as collaborative learning has proved to yield good results, teachers are then advised to let learners learn as either pairs or groups, even if the seating plan makes learners to be scattered from each other [24]. This method of learning collaboratively has been proved by other writers to have some advantages as research argues that in group work learners have an opportunity to learn from each other as they can better unpack some ideas clearer to respective classmates, as compared with how a teacher would have tackled it, this at times resulting from that some learners have an introvert attitude, as such become reluctant to seek assistance from their teachers [25].

From the collected data, it also emerged that the newly designed seating plan for learners to be positioned apart from their peers seemed to have some adverse impacts on learning, and specifically for the purposes of this investigation, isolation led to attitudes of neglect, this having an influence on language development [26]. The current seating plan in the schooling environment is attached with compliance of COVID-19 regulations on social distancing as prescribed by the South African government. Elements of anxiety in learners revolve around logistics encounters with regard to loneliness, discomposure, and loss of peer interaction [27]. To the authors' perspective, these critical aspects are likely to have an influence on academic content and uncertainty in learner progression. Teachers also reported that learners whose comprehension levels were not of the expected average normally raised concerns that the current seating plan causes lots of stress, likely to impact on their development in learning.

On the other side, teachers also reported their concern regarding learner isolation as this might impact on emotional and social learning [28]. Proficiency gaps in reading abilities were noted by language teachers to have erupted ever since the innovations on the classroom seating plans, in so much that isolation even from friends and family in general, have normally resulted into an unhealthy social learning environment. This finding is in line with the views that for a task to be considered as outcomes-based, learners are expected to cultivate some links in between the content learnt and the encompassing learning styles thereof. Moreover, as learners comprehend and intermingle in the target language that is being learnt, there seems to be a crucial need for them to interact and communicate among themselves for easy access to acquire the logistics of the new and the unacquainted language. Engaging in such a strategy is likely to rebuild self-confidence, this leading to improved language proficiency levels [29]. For satisfactory progress, learning in schools needs to be a process administered through social relationships, including oneself, life, as well as collaborating with others through unanimous decision-making [30, 31]. By so doing, the school then becomes a family because of the supportive networking by both teachers and groups of learners. For such networking to be lost, it then really becomes a greatest concern ever. As the authors of this manuscript, we therefore opine for paramount importance that language teachers devise the necessary strategies aimed toward improved language proficiency with revised approaches to help rebuild the lost self-intrinsic inspiration within learners as swept away by isolation logistics.

*Social Distancing Disbanding Learner Groupings: A Case on Language Development DOI: http://dx.doi.org/10.5772/intechopen.104893*


**4. Discussion**

454 Ecosystems Biodiversity

Average # of bands per primer

Diversity Index (DI)

Effective Multiplex Ratio (E)

Correlation coefficient (r)

Marker Index (MI)

Method

RAPD 14.60 0.29 17.13 4.914 Within Female 0.492

**ISSR** 16.20 0.33 16.20 5.289 Within Female 0.606

**DAMD** 19.00 0.23 19.00 4.423 Within Female 0.245

**SLXY** 17.25 0.25 17.25 4.366 Within Female 0.489

Table 3. The comparison of different PCR methods for the assessment of genetic diversity in

Mantel Coefficient for 100 random iterations (Z)

Standard Normal

RAPD / ISSR 21.179\*\* 673.781 0.8432+++ RAPD / DAMD 9.968\*\* 970.869 0.5009+ RAPD / SLXY 18.021\*\* 828.749 0.7853+++ ISSR / DAMD 8.895\*\* 790.384 0.4619+ ISSR / SLXY 17.562\*\* 680.878 0.7854+++ DAMD / SLXY 6.399\*\* 978.962 0.3850+

Table 4. Correlations among the distance matrices for the RAPD, ISSR, DAMD and SLXY primer PCR band data in case of the gender based set of the betelvine landraces. A significant, (p = 0.005), standard normal variate **(g)** was obtained among the matrices

generated by all the four methods in all possible pairs of matrix comparison.

Variate (g)

**\*\*** Highly significant values (Critical value **p**0.005 = 2.575)

Average similarity index (SI)

Comparison

Between Male and

Between Male and

Between Male and

Between Male and

Methods compared Matrix 1 / Matrix 2

**+++**: High correlation

**<sup>+</sup>**: Moderate or low correlation

Within Male 0.544

Female 0.282 Within Male 0.671

Female 0.400 Within Male 0.389

Female 0.183 Within Male 0.545

Female 0.266

the gender set of betelvine landraces.

Dioecy is a widespread condition in flowering plants. Despite their recent evolutionary origin, 6% of the 240,000-angiosperm species are dioecious and 7% of 13,000 genera of angiosperms include dioecious species, suggesting that it has arisen many times during flowering plant evolution (Renner and Ricklefs, 1995). Dioecy is correlated with perennial climbing growth, wind or water pollination and has a preponderence in tropical flora. Natural selection, development of complex physiological and morphological traits, male fitness, ecological context, sex ratio, phylogenetic perspective and nuclear-cytoplasmic gynodioecy are some of the factors affecting distribution of dioecy. The plant taxa can offer insights into level of dimorphism that exists prior to the evolution of complete dioecy and the tradeoffs or constraints faced by hermaphrodites. The betelvine is one such interesting dioecious plant with an almost obligate vegetative propagation, lianaceous habit and perennial growth that provides a good system for studying molecular aspects of dioecy in general and functional dioecy in particular.

The study with PCR profiles was the first step towards resolving gender differences, if any, in betelvines. In an earlier study with 53 landraces that included only a few landraces with known genders, the RAPD method had provided a distinction between male and female betelvines (Verma *et al.*, 2004). In the present study with a larger sampling specifically amongst the betelvines with known genders, the bootstrap NJ tree for the RAPD data clearly differentiated the male and female landraces into two separate broad clusters, thereby supporting our original grouping of the landraces in terms of male and female vines based on known or actual flowering data. Banerjee *et al.* (1999) have reported a similar study of RAPD profile variation in another dioecious species, *Piper longum* L. and have further shown that at least two RAPD fragments were consistently associated with male plants. The clear separation of the betelvines on the basis of gender provides important leads for the identification and development of gender specific primers and probes. This work, however, has an important caveat that the economically most important product of the betelvines are the leaves and leaves of both male and female vines have a market value. Therefore the gender specific detection and or diagnosis will have only an academic value in case of betelvines unlike that in plants such as Papaya (Deputy *et al.*, 2002; Ma *et al.*, 2004). ISSR-PCR has been successfully utilized to distinguish gender or gender-specific markers in dioecious plants like hops and datepalm (Jakse et al. 2008; Younis et al. 2008). In the present

SPAR Profiles for the Assessment of Genetic Diversity Between

mechanisms underlying the variation measured.

now exploit the diversity judiciously.

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 457

very interesting to determine the identity and sequence of genomic regions of betelvines that have resulted in the discrete multibanded profiles even under high PCR stringency. **Comparison of the different methods used to assess the genetic diversity in betelvines:** The gender based set of betelvine landraces was systematically analyzed with four different types of primers. For each set of primers used, the profile data were used for the calculation of the Diversity Index (DI), Effective Multiplex Ratio (E) and Marker Index (MI) and Mean probability (p). These calculations allowed a comparison of the four PCR-methods. The values calculated in each case, are given in Table 3. Parson *et al.* (1997) suggested that differences in the chromosomal location of the three types of markers could influence the diversity assessment. Kojima *et al.* (1998), indicated that in wheat RAPDs were more representative of chromosomal regions enriched in repeated sequences, while ISSRs were related, as RFLPs, to coding sequences. A similar case was seen in case of lentil (Sonnate and Pignone, 2001) where the authors could not find any congruence between the RAPD and ISSR method. Carvalho and Schaal (2001) also obtained different levels of polymorphism in cassava, where the SSR-primed markers showed less polymorphism than the RAPD markers. In their case also grouping of varieties from different geographical habitats varied between the RAPD and ISSR techniques. In absence of any pedigree information about the varieties, we could not address the issue of concordance between the molecular profiling based estimates of genetic similarity and pedigree but, we may expect greater genetic information about genetic similarity from the molecular profiling based estimates in accordance with Russell *et al.* (1997). From their result also it was apparent that the different techniques reveal information about distinct regions of the genome. Moreover, the rate of evolution of the primer target site sequence is most likely different for the two types of markers. So depending on this aspect, the divergence shown amongst the genotypes by the different techniques would also differ. Powell *et al.* (1996) suggested, that any estimation of genetic relationships between individual genotypes was affected by, the number of markers, the distribution of markers in the genome (genome coverage) and the nature of evolutionary

The four methods do not reveal polymorphism within gender set to the same extent. Under our experimental conditions ISSR method was found to have the higher Marker index as well as PIC. This is clearly reflected in the bootstrapped Neighbour Joining tree for the ISSR data where the entire female vines clustered together in one broad group while the male vines were separated in at least three distinct sub-groups. In general during the course of the present study we have observed that the male betelvines are invariably more heterogenous than the female vines. Such a result is of significance for the application of breeding methods for the improvement of betelvines. Unfortunately betelvine as a crop is cultivated by vegetative means ever since it was first domesticated. There have been only sporadic attempts at the improvement of betelvines through controlled process. The present study has resulted in the assessment of range of diversity in the betelvines for a breeder who can

In our study we found that ISSR method, which showed the highest Diversity and Marker Index, can be the method of choice for diversity analysis type of studies, in so far as polymorphism or Marker Index is the criterion. In the present study actually all four methods were almost equally useful for the analysis of the betelvine landraces (MI values in the range 4.366 to 5.289, Table 3). In fact the Mantel Test (distance matrix correlation)

study one ISSR primer each with dinucleotide and trinucleotide motifs and three primers with tetranucleotide motifs were tried in PCR and revealed distinct profiles that were however broadly similar across all the landraces. This result indicated that the ISSR regions were apparently conserved at least in length if not in sequence. The polymorphic bands helped to resolve the NJ tree into two major clusters with two groups of male and female betelvine clearly separating out. DAMD method has not been specifically used for any gender distinction studies in plants. For betelvine landraces, the NJ tree separated the male and female genders of the landraces and at the same time the DAMD method also revealed a relatively greater diversity amongst the male betelvines. The primers based on sequences specific to known X and Y chromosome of a dioecious plant *Silene latifolia* were tested with the dioecious betelvines in the gender set of landraces. Here the expectation was that the X and Y specific primers would reveal clear differences in the PCR profiles of the female and male betelvines respectively. These primers when used singly as well as in combination, however, did not reveal any such discrete profile differences. Surprisingly, these primers actually resulted in RAPD like multibanded profile and hence the band data for this was also scored in the same way as for the other three methods. This kind of study has never been done for any other plant to our knowledge. The conclusion about the most similar or most dissimilar landraces was based on cumulative data for all the SLXY primers. The result is actually interesting. Primarily the results indicate that multiple primer binding sites were present in all the DNAs. If these primers were generating multiple products it would appear that they have no co-relation with gender determining sequences in betelvines. This however is not entirely true since the NJ tree from the cumulative data can be clearly resolved into two major clusters, for the male and female betelvine respectively. Thus the PCR products resulting from these primers seemingly differs between the two genders and since several products are formed. It would appear that these primers are amplifying sequences from more than one gender determinant. Further since primers from both X and Y specific sequences gave multiple amplification products from the both male and female betelvines, our results indicate that gender determination in betelvine may either not be dependent on specific sex chromosomes or if such chromosome do exist male and female betelvines have similar sets of sequences on these chromosomes. Considering that all dioecious plants do not always have heteromorphic and distinct sex chromosomes, on the basis of our results with SLXY primers we infer that betelvine is one of those dioecious plants that lack distinct sex chromosomes.

In a novel approach, the fourth group of primers used was actually a heterologous set of primers were derived from X and Y chromosome specific sequences of the dioecious plant *Siliene latifolia*. The use of such primers for the dioecious betelvines was expected to reveal specific information about chromosomal basis for dioecy, if any. Interestingly, the SLXY primers resulted in multibanded profiles of several distinct bands even under stringent PCR conditions clearly indicating that several dispersed sequences homologous to the primers used were present. Though the primers collectively generated data that segregated the male and female vines, no single primer gave a sharply defined dimorphic profile for the two genders. This observation leads us to the conclusion that dioecy in betelvine may not follow the same chromosomal basis as in the case of *Silene latifolia*. Of course it is possible that such a lack of dimorphic profile could also be attributed to lack of strong homology of the primers to the appropriate regions of the betelvine. In this situation however, it would be

study one ISSR primer each with dinucleotide and trinucleotide motifs and three primers with tetranucleotide motifs were tried in PCR and revealed distinct profiles that were however broadly similar across all the landraces. This result indicated that the ISSR regions were apparently conserved at least in length if not in sequence. The polymorphic bands helped to resolve the NJ tree into two major clusters with two groups of male and female betelvine clearly separating out. DAMD method has not been specifically used for any gender distinction studies in plants. For betelvine landraces, the NJ tree separated the male and female genders of the landraces and at the same time the DAMD method also revealed a relatively greater diversity amongst the male betelvines. The primers based on sequences specific to known X and Y chromosome of a dioecious plant *Silene latifolia* were tested with the dioecious betelvines in the gender set of landraces. Here the expectation was that the X and Y specific primers would reveal clear differences in the PCR profiles of the female and male betelvines respectively. These primers when used singly as well as in combination, however, did not reveal any such discrete profile differences. Surprisingly, these primers actually resulted in RAPD like multibanded profile and hence the band data for this was also scored in the same way as for the other three methods. This kind of study has never been done for any other plant to our knowledge. The conclusion about the most similar or most dissimilar landraces was based on cumulative data for all the SLXY primers. The result is actually interesting. Primarily the results indicate that multiple primer binding sites were present in all the DNAs. If these primers were generating multiple products it would appear that they have no co-relation with gender determining sequences in betelvines. This however is not entirely true since the NJ tree from the cumulative data can be clearly resolved into two major clusters, for the male and female betelvine respectively. Thus the PCR products resulting from these primers seemingly differs between the two genders and since several products are formed. It would appear that these primers are amplifying sequences from more than one gender determinant. Further since primers from both X and Y specific sequences gave multiple amplification products from the both male and female betelvines, our results indicate that gender determination in betelvine may either not be dependent on specific sex chromosomes or if such chromosome do exist male and female betelvines have similar sets of sequences on these chromosomes. Considering that all dioecious plants do not always have heteromorphic and distinct sex chromosomes, on the basis of our results with SLXY primers we infer that betelvine is one of those dioecious

In a novel approach, the fourth group of primers used was actually a heterologous set of primers were derived from X and Y chromosome specific sequences of the dioecious plant *Siliene latifolia*. The use of such primers for the dioecious betelvines was expected to reveal specific information about chromosomal basis for dioecy, if any. Interestingly, the SLXY primers resulted in multibanded profiles of several distinct bands even under stringent PCR conditions clearly indicating that several dispersed sequences homologous to the primers used were present. Though the primers collectively generated data that segregated the male and female vines, no single primer gave a sharply defined dimorphic profile for the two genders. This observation leads us to the conclusion that dioecy in betelvine may not follow the same chromosomal basis as in the case of *Silene latifolia*. Of course it is possible that such a lack of dimorphic profile could also be attributed to lack of strong homology of the primers to the appropriate regions of the betelvine. In this situation however, it would be

plants that lack distinct sex chromosomes.

very interesting to determine the identity and sequence of genomic regions of betelvines that have resulted in the discrete multibanded profiles even under high PCR stringency.

**Comparison of the different methods used to assess the genetic diversity in betelvines:** The gender based set of betelvine landraces was systematically analyzed with four different types of primers. For each set of primers used, the profile data were used for the calculation of the Diversity Index (DI), Effective Multiplex Ratio (E) and Marker Index (MI) and Mean probability (p). These calculations allowed a comparison of the four PCR-methods. The values calculated in each case, are given in Table 3. Parson *et al.* (1997) suggested that differences in the chromosomal location of the three types of markers could influence the diversity assessment. Kojima *et al.* (1998), indicated that in wheat RAPDs were more representative of chromosomal regions enriched in repeated sequences, while ISSRs were related, as RFLPs, to coding sequences. A similar case was seen in case of lentil (Sonnate and Pignone, 2001) where the authors could not find any congruence between the RAPD and ISSR method. Carvalho and Schaal (2001) also obtained different levels of polymorphism in cassava, where the SSR-primed markers showed less polymorphism than the RAPD markers. In their case also grouping of varieties from different geographical habitats varied between the RAPD and ISSR techniques. In absence of any pedigree information about the varieties, we could not address the issue of concordance between the molecular profiling based estimates of genetic similarity and pedigree but, we may expect greater genetic information about genetic similarity from the molecular profiling based estimates in accordance with Russell *et al.* (1997). From their result also it was apparent that the different techniques reveal information about distinct regions of the genome. Moreover, the rate of evolution of the primer target site sequence is most likely different for the two types of markers. So depending on this aspect, the divergence shown amongst the genotypes by the different techniques would also differ. Powell *et al.* (1996) suggested, that any estimation of genetic relationships between individual genotypes was affected by, the number of markers, the distribution of markers in the genome (genome coverage) and the nature of evolutionary mechanisms underlying the variation measured.

The four methods do not reveal polymorphism within gender set to the same extent. Under our experimental conditions ISSR method was found to have the higher Marker index as well as PIC. This is clearly reflected in the bootstrapped Neighbour Joining tree for the ISSR data where the entire female vines clustered together in one broad group while the male vines were separated in at least three distinct sub-groups. In general during the course of the present study we have observed that the male betelvines are invariably more heterogenous than the female vines. Such a result is of significance for the application of breeding methods for the improvement of betelvines. Unfortunately betelvine as a crop is cultivated by vegetative means ever since it was first domesticated. There have been only sporadic attempts at the improvement of betelvines through controlled process. The present study has resulted in the assessment of range of diversity in the betelvines for a breeder who can now exploit the diversity judiciously.

In our study we found that ISSR method, which showed the highest Diversity and Marker Index, can be the method of choice for diversity analysis type of studies, in so far as polymorphism or Marker Index is the criterion. In the present study actually all four methods were almost equally useful for the analysis of the betelvine landraces (MI values in the range 4.366 to 5.289, Table 3). In fact the Mantel Test (distance matrix correlation)

SPAR Profiles for the Assessment of Genetic Diversity Between

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 459

Fig. 4. The NJ tree for the cumulative band data for all the four PCR methods after 1000 replicate bootstrap analysis clearly depicts the separation of the out group taxon, *Piper hamiltonii* (501) from the betelvines which in turn are clearly distributed into separate clusters of male and female landraces. Interestingly each cluster of male or female landraces is further resolved into two sub clusters each, demarked in the figure with smaller labeled parenthesis. The scale at the bottom of the figure is for the Jaccard coefficients. Each landrace is identified by its number and color code as in Table 1. The dashed line through

the figure separates the NJ tree into the respective gender halves.

analysis (Table 4) also resulted in highly significant normal standard variate g > p0.005 = 2.575 in all cases. However, here RAPD and ISSR have resolved the landraces almost equally similarly since the correlation (r = 0.8432, Table 4) was strong between the distance matrices of the two methods. This is an interesting observation and it is suggestive of the possibility that the landraces have more differences amongst themselves in their minisatellite and related tandem repeat sequences. This is actually supported by the observation that all DAMD bands in betelvine were polymorphic.

Sex detemination systems based on heteromorphic X and Y sex chromosomes are particularly interesting to study from both a developmental and evolutionary perspective. There are many paralleles between the sex chromosomes, in different species even between animals and plants. The evolution of heteromorphic sex chromosome systems in widely differing species suggests that similar forces may have been involved in each case (Charlesworh, 1992; Ellis, 1998; Charlesworth and Guttman, 1999). The *Silene* genus is an example of how the evolution of an XY system contributes to morphological change and speciation. The Y chromosome differs from all other chromosomes not only in that it is the only chromosome that does not recombine along majority of its length, but also is being present only in the male sex in a permanent haploid condition (Y genetic isolation), in having a common ancestry and persistent meiotic relationship with the X, and the tendency of its genes to degenerate during evolution (Y genetic erosion).  The functional coherence of the Y can be achieved relatively early during Y evolution, which might be an essential condition for the maintenance of an XY system. Filatov *et al.* (2001) found several differences in polymorphism of the regions of X and Y chromosomes. In another study done by Lebel-Hardenack *et al.* (2002) for genetically mapping of the sexdetermination loci on the male-specific Y chromosome, it was found that *S.latifolia* has three dispersed male-determining loci on the Y chrosome. The sorrel *Rumex acetosa* (Polygonaceae) is a perennial dioecious weed, which possesses sex chromosomes (XX in females, XY1 Y2 in males). These studies indicated that it is only in the recent years that some details are emerging about molecular mechanisms and profiles vis-à-vis gender discrimination in plants.

The four methods tested with the gender set of betelvines have resulted in separation of the male and female betelvines. Though the individual sub-clusters in the four methods are not congruent however the primary separation of the male and female betelvines is clear and unambiguous. Thus the gender distinction of the betelvine landraces in terms of flowering is strongly supported by molecular profiling with four different PCR methods. This is an important and an interesting result. The four methods result in discrete profiles that reflect different genomic regions and in spite of that, the methods allow the landraces to be segregated on the basis of gender. Thus, it appears that there are several levels of genomic differences between the male and female betelvines. In other words, it appears that gender distinction in betelvine may not be confirmed to a few chromosomes or chromosomal regions. This inference is actually supported by lack of male specific and female specific PCR profiles when Y and X chromosome specific primers were respectively used. This further strengthens our inference that dioecy in betelvines is not apparently determined by distinct heteromorphic sex chromosomes. In order to have a comprehensive distribution of landraces, the data generated by all four methods combined and considered cumulatively.

analysis (Table 4) also resulted in highly significant normal standard variate g > p0.005 = 2.575 in all cases. However, here RAPD and ISSR have resolved the landraces almost equally similarly since the correlation (r = 0.8432, Table 4) was strong between the distance matrices of the two methods. This is an interesting observation and it is suggestive of the possibility that the landraces have more differences amongst themselves in their minisatellite and related tandem repeat sequences. This is actually supported by the observation that all

Sex detemination systems based on heteromorphic X and Y sex chromosomes are particularly interesting to study from both a developmental and evolutionary perspective. There are many paralleles between the sex chromosomes, in different species even between animals and plants. The evolution of heteromorphic sex chromosome systems in widely differing species suggests that similar forces may have been involved in each case (Charlesworh, 1992; Ellis, 1998; Charlesworth and Guttman, 1999). The *Silene* genus is an example of how the evolution of an XY system contributes to morphological change and speciation. The Y chromosome differs from all other chromosomes not only in that it is the only chromosome that does not recombine along majority of its length, but also is being present only in the male sex in a permanent haploid condition (Y genetic isolation), in having a common ancestry and persistent meiotic relationship with the X, and the tendency of its genes to degenerate during evolution (Y genetic erosion).  The functional coherence of the Y can be achieved relatively early during Y evolution, which might be an essential condition for the maintenance of an XY system. Filatov *et al.* (2001) found several differences in polymorphism of the regions of X and Y chromosomes. In another study done by Lebel-Hardenack *et al.* (2002) for genetically mapping of the sexdetermination loci on the male-specific Y chromosome, it was found that *S.latifolia* has three dispersed male-determining loci on the Y chrosome. The sorrel *Rumex acetosa* (Polygonaceae) is a perennial dioecious weed, which possesses sex chromosomes (XX in females, XY1 Y2 in males). These studies indicated that it is only in the recent years that some details are emerging about molecular mechanisms and profiles vis-à-vis gender

The four methods tested with the gender set of betelvines have resulted in separation of the male and female betelvines. Though the individual sub-clusters in the four methods are not congruent however the primary separation of the male and female betelvines is clear and unambiguous. Thus the gender distinction of the betelvine landraces in terms of flowering is strongly supported by molecular profiling with four different PCR methods. This is an important and an interesting result. The four methods result in discrete profiles that reflect different genomic regions and in spite of that, the methods allow the landraces to be segregated on the basis of gender. Thus, it appears that there are several levels of genomic differences between the male and female betelvines. In other words, it appears that gender distinction in betelvine may not be confirmed to a few chromosomes or chromosomal regions. This inference is actually supported by lack of male specific and female specific PCR profiles when Y and X chromosome specific primers were respectively used. This further strengthens our inference that dioecy in betelvines is not apparently determined by distinct heteromorphic sex chromosomes. In order to have a comprehensive distribution of landraces, the data generated by all four methods

DAMD bands in betelvine were polymorphic.

discrimination in plants.

combined and considered cumulatively.

Fig. 4. The NJ tree for the cumulative band data for all the four PCR methods after 1000 replicate bootstrap analysis clearly depicts the separation of the out group taxon, *Piper hamiltonii* (501) from the betelvines which in turn are clearly distributed into separate clusters of male and female landraces. Interestingly each cluster of male or female landraces is further resolved into two sub clusters each, demarked in the figure with smaller labeled parenthesis. The scale at the bottom of the figure is for the Jaccard coefficients. Each landrace is identified by its number and color code as in Table 1. The dashed line through the figure separates the NJ tree into the respective gender halves.

SPAR Profiles for the Assessment of Genetic Diversity Between

economically important plant part is the leaf.

*longum* L. *Current Science* 77(5): 693-695.

markers. *Euphytica* 120: 133-142.

*Genet.* 106: 107-11.

388-390.

Scientific Publishers, Oxford pp. 25-50.

**5. References**

Male and Female Landraces of the Dioecious Betelvine Plant (*Piper betle* L.) 461

The NJ tree from such a combined data for all methods is given in Figure 4. This tree also clearly separates the male and female betelvines. Interestingly the tree can be resolved into two subclusters within both male and female clusters as in the figure. MA, MB, FA and FB are the subclusters for males and females betelvines respectively (illustrated best by a radial tree form of the NJ tree as shown in Figure 5). We infer possibility of having at least two distinct ancient lineages for the male and female betelvines. In the absence of historical data and chronology of cultivation of these landraces it is not possible to confirm that there were actually a few discrete lineages of the cultivated betelvines and that the present day betelvine landraces are descendants of these lineages. The primary interest in all these studies is fuelled by the fact that dioecy as a proportion of accounts for only a small fraction of the numbers of flowering plants, yet in distribution across the plant families it is wide. Further, at the applied level, for plants of economic importance, detection and diagnosis of plant sex as an early event is desirable when the economically viable and important plant part is gender associated such as for example the fruits. In this context, betelvine is an exception in that for both male and female plants, the

Abe J., Xu D. H., Suzuki Y., Kanazawa A. and Shimamoto Y. (2003). Soybean germplasm pools in Asia revealed by nuclear SSRs. *Theor. Appl. Genet.* 106: 445-453. Ajibade S. R., Weeden N. F. and Chite S. M. (2000). Inter simple sequence repeat analysis of

Banerjee N. S., Manoj P. and Das M. R. (1999) Male sex-associated RAPD markers in *Piper*

Bhattacharya E. and Ranade S. A. (2001). Molecular distinction amongst varieties of

Carvalho L. J. C. B. and Schaal B. A. (2001). Assessing genetic diversity in the cassava

Charlesworth D. and Guttman D. S. (1999). The evolution of dioecy and plant sex

deKochko A. and Hamon S. (1990). A rapid and efficient method for the isolation of restrictable total DNA from plants of genus *Abelmoschus*. *Plant Mol. Bio. Rep.* 8: 3-7. Deputy J. C., Ming R., Ma H., Liu Z., Fitch M. M., Wang M., Manshardt R., Stiles J.I. (2002)

Filatov D. A., Moneger F., Negrutiu I. and Charlesworth D. (2000). Low variability in a Y-

(*Manihot esculenta* Crantz) germplasm collection in Brazil using PCR-based

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Fig. 5. The radial NJ tree for the cumulative band data for all the four PCR methods after 1000 replicate bootstrap analysis clearly depicts the separation of the out group taxon, *Piper hamiltonii* (501) from the betelvines which in turn are clearly distributed into separate clusters of male and female landraces. Each cluster of male or female landraces is very distinctly resolved into two sub clusters each. The scale at the bottom of the figure is for the Jaccard coefficients. Each landrace is identified by its number and color code as in Table 1. The dashed line through the figure separates the NJ tree into the respective gender halves.

The NJ tree from such a combined data for all methods is given in Figure 4. This tree also clearly separates the male and female betelvines. Interestingly the tree can be resolved into two subclusters within both male and female clusters as in the figure. MA, MB, FA and FB are the subclusters for males and females betelvines respectively (illustrated best by a radial tree form of the NJ tree as shown in Figure 5). We infer possibility of having at least two distinct ancient lineages for the male and female betelvines. In the absence of historical data and chronology of cultivation of these landraces it is not possible to confirm that there were actually a few discrete lineages of the cultivated betelvines and that the present day betelvine landraces are descendants of these lineages. The primary interest in all these studies is fuelled by the fact that dioecy as a proportion of accounts for only a small fraction of the numbers of flowering plants, yet in distribution across the plant families it is wide. Further, at the applied level, for plants of economic importance, detection and diagnosis of plant sex as an early event is desirable when the economically viable and important plant part is gender associated such as for example the fruits. In this context, betelvine is an exception in that for both male and female plants, the economically important plant part is the leaf.