**9. Extracellular Vesicles (EVs)**

Extracellular vesicles (EVs) are mediators that can modify the function of target cells by transferring proteins and genomic materials to other cells; thus, EVs have an active participation in cell-to-cell communication [171]. EVs shred from a variety of cells and have a number of important physiological as well as pathological functions as they are capable of tra fficking and transfecting the genetic material from cell to cell. The biogenesis and contents of EVs predominantly depends on the originating cell type and their surrounding microenvironment [172]. Several studies using electron microscopy analysis to characterize the morphology of EVs demonstrate that are spherical with lipid bilayer membrane [173,174]. The correct classification of EVs still a manner of debate and the majority of the information in the literature classify them according to their size and the di fferent biogenesis pathways. Typically, EVs are categorized as exosomes (~40–100 nm), microvesicles (~100–1000 nm) and apoptotic bodies (~1000–5000 nm) based on their size and origin. Microvesicles and apoptotic bodies are formed directly via budding of the plasma membrane, whereas exosomes are produced via an endocytic pathway [174]. Distinction between di fferent EVs subgroups is di fficult, due to the minimal physical and morphological di fferences, to the lack of specific markers, and to the fact that the same cellular source may dynamically produce di fferent class of EVs in response to di fferent conditions [175]. Recently, the international society of extracellular vesicles has recommended classifying the vesicles according to the their size in small EVs (<100–200 nm) and medium/large EVs (>200 nm), or density (low, middle, high, with each range will defined) or their biochemical composition (e.g., CD63+ve) [176]. Currently, there is no single method allowing for accurate characterization and discrimination of the di fferent EVs classes [175]. EVs can be ordinarily isolated from di fferent biological fluids using the di fferential and buoyant density centrifugation methods followed by ultrafiltration/size exclusion chromatography or flow cytometry or precipitation using polymers or antibodies to enrich the pure EVs population [177]. EVs play an important role in cell-to-cell communication and influence a variety of cellular functions, including cytokine production modulation, cell proliferation, apoptosis and metabolism, by transferring their protein, lipid or messenger RNA and micro RNA molecules [173,178]. EVs can be isolated both biological fluids (e.g., plasma/serum, urine, cerebrospinal fluid, saliva, etc.) and in vitro from cell-conditioned media. Moreover, as EVs are natural carriers of bioactive molecules, di fferent research studies are addressing the therapeutic potential of EVs due to their specific genetic material packaging capabilities [175]. Several groups have identified EVs in maternal biofluids during normal and complication of pregnancies and the potential role of EV during pregnancy have been reviewed in details by our group previously [179–182].

#### **10. Extracellular Vesicles**/**Exosomes in Normal and Preeclamptic Pregnancies**

Di fferent research studies utilized a variety of experimental models (i.e., biological fluids, primary placental trophoblasts, trophoblast cell line, placental explant, placental perfusate etc.) to isolate di fferent subpopulations (exosomes, microvesicles) of EVs and studied their role in the context of healthy as well as in pathological pregnancies. Small and large EVs originating from the placenta have been identified in maternal plasma. Concentrations of both total and placenta-derived exosomes present in maternal circulation increase across gestation [183] and are higher in complicated pregnancies (such as those a ffected by PE) as compared to normal pregnancies [184,185]. Interestingly, the global miRNA profile within small vesicles such as exosomes di ffers between normal and PE pregnancies across gestation and it is likely that PE is not only associated with changes in the circulating levels of exosomes, but also in their miRNA content [186]. Recently, Biro et al. identified that hsa-miR-210

level increased in the circulating exosomes isolated from PE pregnancies [187]. Poor placentation is associated with hypoxia and oxidative stress, which are features of PE and a ffects the invasion of extravillous trophoblast (EVT) and the uterine spiral arterial remodeling. Truong et al. studied whether low oxygen tension alters exosome release and the exosomal miRNA profile from HTR-8/SVneo cell line and examined their interaction with endothelial cells [188]. HTR-8/SVneo cells are commonly used as a model for EVT cells, although they are not ideal, as they contain a heterogenous population of trophoblast and stromal cells [189]. In this study, low oxygen tension to exosomes from EVTs cultured under normoxic conditions. Moreover, a specific set of miRNAs within exosomes from EVTs cultured under hypoxia were identified, and these miRNAs are present in circulating exosomes at early gestation from women who develop PE later in pregnancy. This data suggests that aberrant extracellular vesicle signaling is one of the common factors in the development of PE. In normal healthy pregnancy, syncytiotrophoblast derived EVs release into the maternal blood stream where they act upon their target endothelial cells and circulating immune cells [33,190–192]. Placental EVs carry di fferent proteins, lipids and nucleic acids that play a crucial role in feto-maternal communication to maintain pregnancy [193]. Interestingly, concentrations of large EVs gradually increase through pregnancy irrespective of their origin [186] and these EVs convey pro-inflammatory and pro-thrombotic antigens that might contribute to the hypercoagulable state observed in the last trimester of pregnancy [186]. Chang et al. identified that high levels of preeclamptic exosomes contain abundant sFlt-1 and sEng that can induce vascular dysfunction as these proteins were captured by vascular endothelial cells [194]. Tannetta et al. investigated the level of expression of placental protein 13 in syncytiotrophoblast derived extracellular vesicles (STBEVs) isolated from PE and normal pregnancy placental perfusate and found it was low in PE placenta [195]. Tong et al. described a novel mechanism by which placental EVs can attenuate PE pathogenesis in the presence of antiphospholipid antibody (aPL), which can induce the synthesis of toll-like receptors on placental EVs to increase the level of expression of mitochondrial DNA in these vesicles [196]. Thus, placenta-derived EVs are involved in gene regulation, placental homeostasis and cellular function that overall reflect the placental-maternal crosstalk [197]. Placental exosomes were also observed in fetal blood and their concentration correlated with fetal growth [198]. The concentration of placental exosomes in the fetal circulation was higher than that found in the maternal circulation and was also higher in pregnancies a ffected by PE [199]. Interestingly, not only the concentration of circulation exosomes in PE is di fferent compared with normal pregnancies, and specific changes in the protein cargo of exosomes in PE have been identified [200].

Another recent study measured the level of di fferent biomarkers including copeptin, annexin V and placental growth factor in maternal serum derived microparticles at 10–14 gestational weeks in women with PE and compared with that of normal healthy pregnancy [201]. Interestingly, the levels of nitric oxide synthase enzyme in the STBEVs were lower in STBEVs from PE compared to normal pregnancies [202]. In this regard, in a similar study, the levels of the protein neprilysin were increased in EVs of PE placenta [203].

Kohli et al. identified a novel pathway by which the placental EVs interact and causes release of EVs from endothelial cells and platelets that further activate the inflammasome in the trophoblast resulting in the development of PE [204]. The role of EVs in relation to PE pathophysiology including their di fferent contents has been summarized in Table 2.

**Table 2.** Updated Research Studies on EVs in PE Pathophysiology.

**Table 2.** *Cont.*

A number of drugs that can be used to treat PE appear to modulate EV expression. Some studies also addressed the mode of action of different antihypertensives, including thiazide diuretics that are used to treat the hypertension in PE. Hu et al. identified some changes in the sodium transporters in the renal tubule that were incorporated in the urinary exosomes isolated from PE women [206]. Another very interesting study by Chamley L. et al. identified melatonin as an effective agent that can reduce the endothelial cell activating placental EVs release in PE [209]. In a similar study, transthyretin which is the thyroxin binding protein, was found in aggregated form and packaged in the small placental EVs in PE [210]. Xu et al. identified potential molecular mechanisms by which vitamin-D can reduce oxidative-stress induced PE [213]. Among the different therapeutic agents, the efficacy of aspirin was evaluated due to its availability and cost-effectiveness. However, there is lack of understanding in the mechanism of action of aspirin in the context of EV secretion regulation.

#### **11. E**ff**ects of LDA on Exosomal Secretion**

Up until now, there has been very limited evidence on the potential effect of aspirin on the release and content of EVs. Goetzl E.J. et al., discovered that in the presence of some coagulation factors (e.g., thrombin/ collagen) induces changes in the plasmatic levels of platelet-derived exosomes and their protein content (i.e., α-granule chemokines CXCL4 and CXCL7 and cytoplasmic high-mobility group box 1 (HMGB1)) [214]. Incubation of normal platelets with aspirin significantly inhibits arachidonic acid (AA)-induced platelet reactivity, EV formation and pro-coagulant activity [215]. Interestingly, aspirin therapy can significantly reduce microparticle (MP) shredding from erythrocytes, monocytes and vascular smooth muscle cells, reversing the effects of diabetes-induced stress on these cells [216]. Other studies have identified that aspirin changes the miRNA profile and EV release from platelet [217]. Syncytiotropholast-derived extracellular vesicles that are placental alkaline phosphatase (PLAP) positive inhibit the aggregation of platelets that were treated with aspirin [218]. Tannetta et al. observed that STBEV that are placental alkaline phosphatase (PLAP) positive inhibit the aggregation of platelets that were treated with aspirin [218]. On the contrary, platelets were activated and thrombus formation was increased by the STBEV isolated from preeclamptic placentas. Another study observed the effect of anticoagulant therapy (treatment with either unfractionated heparin (UFH) or low molecular weight heparin (LMWH), and/or LDA) on cell derived microparticles and outcome of pregnancy [219]. These findings indicate that placenta-derived extracellular vesicles may provide understanding in their potential role in low dose aspirin induced placental functions. Although there is significant evidence for dysregulation of both concentrations and bioactivity of circulating placental EVs in PE compared to normal pregnancies, no studies have described the potential effect of aspirin on EVs released from placental cells and their bioactivity.


Meet the editor

Montaha Bouezzeddine received a DiplEng in Networks and Communication Systems, a master's in Engineering Research (I-MARS) from INSA Rennes, France, in 2012, and a Ph.D. in Electrical Engineering from the University of Duisburg-Essen, Germany, in 2018. From 2012 to 2017, she worked as a research engineer at RheinMain University of Applied Sciences, Germany, on the design of multiport antenna systems for mobile com-

munications. Between 2017 and 2019, she worked at Telit on the design of Bluetooth and BLE hardware, then at Kathrein Automotive (now, Continental) on the design of antennas for automotive applications and at Ford Company as a technical consultant. In October 2019, she joined Fraunhofer IZM where she is currently a research engineer. Her research interests include MIMO antennas, antenna arrays, millimeter-waves, tunable and reconfigurable antennas, characteristic modes theory, antennas for automotive applications, IoT, and adaptive tuning and digital control. Dr. Bouezzeddine was the recipient of the ESoA Best Student Paper Award at EU-CAP 2016 and received honorary awards in the 2016 student Feko competition and RADIO conference 2016. She is also a reviewer for several international journals.

Contents

Project-Based Learning *by Andrés Díaz Lantada*

Teaching IIoT through Hands-on Activities *by Gustavo Sanchez and Devika Kataria*

*by John Mitchell and David Guile*

and Everyday Life Experiences

*by William Deng Tap*

Education *by Pritpal Singh*

*by Linda Vanasupa and Gilda Barabino*

**Preface XI**

**Chapter 1 1**

**Chapter 2 19**

**Chapter 3 37**

**Chapter 4 53**

**Chapter 5 77**

**Chapter 6 101**

**Chapter 7 119**

Using ICT and Energy Technologies for Improving Global Engineering

Teaching and Learning Mathematics for Understanding, Enjoyment

The Multi-Tier Instrument in the Area of Chemistry and Science *by Habiddin Habiddin and Septiana Ayuningrum Nofinadya*

Fusion Skills and Industry 5.0: Conceptions and Challenges

An Engineering Education of Holism: Einstein's Imperative

Engineering Education 5.0: Strategies for a Successful Transformative


**1. Introduction**

82 Cellulose – Medical, Pharmaceutical and Electronic Applications

Bioelectronics 25: 2343–2350

518-527

1961

15: S210–S214

61(12):1020–32

and Bioelectronics 26(5): 2742–5

Letters 64 (22): 2427–2430

RRL 3 (9): 308– 310

Technol. 6: 332-335

[46] Zhao X, Lu X, Tze W.T.Y and Wang P (2010) A single carbon ber microelectrode with branching carbon nanotubes for bioelectrochemical processes. Biosensors and

[47] Sungryul Yun S, and Kim J (2011) Mechanical, electrical, piezoelectric and electro-active behavior of aligned multi-walled carbon nanotube/cellulose composites. Carbon 49:

[48] Martins R, Barquinha P, Pereira L, Correia N, Gonçalves G, Ferreira I, and Fortunato E (2009) Selective floating gate non-volatile paper memory transístor. Phys. Status Solidi

[49] Ferreira I, Brás B, Correia N, Barquinha P, Fortunato E, and Martins R (2010) Self-Rechargeable Paper Thin-Film Batteries: Performance and Applications. J. Display

[50] Liangbing Hu L, Choi J.W, Yang Y, Jeong S, Mantia F, Cui L-F, and Cui Y (2009) Highly

[51] Tobjörk D, and Ronald Österbacka R (2011) Paper Electronics. Adv. Mater. 23: 1935–

[52] Pushparaj V.L, Shaijumon M.M, Kumar A, Murugesan S, Ci L, Robert Vajtai R, Linhardt R.J, Nalamasu O, and Ajayan P.M (2007) Flexible energy storage devices based on

[53] Lee K.B (2005) Urine-activated paper batteries for biosystems. J. Micromech. Microeng.

[54] Baptista A.C, Martins J.I, Fortunato E, Martins R, Borges J.P, and Ferreira I (2011) Thin and flexible bio-batteries made of electrospun cellulose-based membranes. Biosensors

[55] Lee KY, Jeong L, Kang YO, Lee S.J, and Park W.H (2009) Electrospinning of polysaccharides for regenerative medicine. Advanced Drug Delivery Review

[56] Sill T.J, and von Recum H.A (2008) Electrospinning: Applications in drug delivery and

[57] Penchev H, Paneva D, Manolova N, and Rashkov I (2010) Hybrid nanofibrous yarns based on N-carboxyethylchitosan and silver nanoparticles with antibacterial activity prepared by self-bundling electrospinning. Carbohydrate Research 345(16):2374–80 [58] Li J, Vadahanambi S, Kee C-D, and Oh I-K (2011) Electrospun fullerenol-cellulose

[59] Shuiping L, Lianjiang T, Weili H, Xiaoqiang L, and Yanmo C (2010) Cellulose acetate nanofibers with photochromic property: Fabrication and characterization. Materials

[60] Shari N, Tajabadi F, and Taghavinia N (2010) Nanostructured silver bers: Facile synthesis based on natural cellulose and application to graphite composite electrode for

oxygen reduction. International Journal of hydrogen energy 35: 3258-3262

conductive paper for energy-storage devices. PNAS 106(51): 21490–21494

nanocomposite paper. PNAS 104 (34): 13574–13577

tissue engineering. Biomaterials 29(13): 1989–2006

biocompatible actuators. Biomacromolecules 12(6):2048–54

A biomaterial is defined as a material, either man-made or natural, intended to interact with biological systems. It does not have a chemical effect in the organism, nor thus it need to be metabolised to be active like for example drugs 1. When inserted into the body, a local tissue inflammatory reaction called foreign body reaction is induced 2. This reaction may either favour or adversely affect the tissue repair process.

Cellulose and its derivatives are well tolerated by most tissues and cells 3-5. These nontoxic materials have good biocompability, therefore, they offer several possibilities in medical applications. Regenerated cellulose sponges have also been used in experimental surgery for decades as it does not affect the healing process, but acts as a chemoattractant inducing cells involved in the repair process to migrate towards it 6-8.

We have studied different biomaterials including cellulose in search for an optimal bone substitute. In bone defects, regenerated cellulose supported with cotton fibres was shown to allow new bone in-growth to some degree 9-11. Oxidation with periodate and hydrogen peroxide, or carbamination further improved its biocompability but not enough to be used as bone substitutes. We also expected to increase the osteostimulating property of regenerated cellulose by coating it with a silica-rich hydroxyapatite (HA) as it resembles the mineral composition of bone. To our disappointment, the HA-coated cellulose did not promote bone formation but favoured instead inflammation and fibroplasia. Since the bone implant study revealed unexpectedly an enormous ability of the HA-implants to induce granulation tissue, the coated cellulose was tested subcutaneously as well. These studies showed that the HA-coated cellulose not only attracted inflammatory cells but also bone marrow-derived progenitor cells of both haematopoietic and mesenchymal origin (see box 1). In this chapter, we will discuss cellulose as implant material with emphasis on the cell guiding properties of regenerated cellulose coated with silica-rich HA.

© 2013 Ekholm et al., licensee InTech. This is an open access chapter 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. © 2013 Ekholm et al., licensee InTech. This is a paper 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.


**2. Importance of** *Streptomyces*

Streptomycetes are bacteria that produce the largest amount of commercially used antibiotics worldwide. To date, many *Streptomyces* genomes have been sequenced, and any approach that offers the possibility of increasing yields must be analyzed to increase production levels or produce more effective compounds against bacterial infections. Currently, there is great interest in the isolation of new *Streptomyces* strains from unusual environments as new sources of antibiotics [8].

Many antibiotics have precursors that act as intermediates for different metabolic pathways. Gunnarsson et al. (2004) described how central carbon metabolism is linked to producing many different antibiotics [9]; however, little has been achieved to improve the synthesis of inermediates and influence the biosynthesis of antibiotics or other commercially important compounds.

Genome sequencing of important antibiotic-producing *Streptomyces* has allowed the analysis of genes that encode the enzymes involved in carbon metabolism pathways. Gene multiplicity exists in the genomes of these organisms and up to four copies of the same gene can be found; however, the relevance of this fact has not been established. Gene multiplicity or redundancy is very common in the chromosomes of many microorganisms, especially *Streptomyces*.

## **3. Gene multiplicity in glycolytic pathway genes**

There are many databases, such as KEGG, to identify how many genes code for the same activity. It is known that nine enzymes participate in the glycolytic pathway, as shown in **Table 1**, and it was found that three genes encode phosphofructokinase, which catalyzes the phosphorylation of fructose 6-phosphate in most antibiotic-producing *Streptomyces* species. Only *S. coelicolor* and *S. venezuelae* have two genes.

The conversion of fructose 6-phosphate to fructose 1,6-bisphosphate with the concomitant hydrolysis of adenosine triphosphate represents the first irreversible step specific to glycolysis. This reaction catalyzed by phosphofructokinase (PFK; EC <IP_ADDRESS>) is subjected to tight control, thus rendering it a critical regulatory point of the glycolytic flux [10]. The genes that encode PFK present a high multiplicity, and in these 11 antibiotic-producing *Streptomyces*, 31 proteins have been noted. The PFKs have amino acids between 341 and 345, and only *S. griseus* and *S. venezuelae* have two copies, whereas the remainder has three genes. The 31 proteins have a very high identity with some identical regions along the sequence, and at the aminoterminal end, a highly conserved domain GGDCPGLNAVIR is present; 133 residues out of 350 are fully conserved, representing 38% identity. Despite the high resemblance, the phylogenetic tree is divided into two clades, one of which is split into two subgroups, as shown in the tree, each copy is distributed in one clade, and two of them are more closely related to each other. This clade includes *S. griseus* and *S. venezuelae*, which have only two copies of the PFK-coding genes, suggesting that retention of a third gene copy has not occurred in these species (**Figure 1**). Unlike *Escherichia coli*, which has two PFKs that do not have a common ancestor, *Streptomyces* does [11].

*Actinobacteria - Diversity, Applications and Medical Aspects*

#### **Table 1.**

*Gene multiplicity in glycolytic pathway genes.*

### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

#### **Figure 1.**

*Partial multiple alignments of fosfofructokinase proteins from antibiotic producing* Streptomyces *(A) and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

*Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

The next reaction is performed using aldolase (EC <IP_ADDRESS>), which catalyzes the conversion of fructose 1-6-diphosphate to glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. Most *Streptomyces* have two genes that code for this enzyme, except for *S. lavendulae*, which has four genes. The SLAV\_00910 and SLAV\_38480 proteins have 100% identity; therefore, are coded by duplicated genes.

Triosephosphate isomerase (EC <IP_ADDRESS>) is an enzyme that converts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Of all the antibiotic-producing *Streptomyces*, only *S. coelicolor* had two proteins for this activity with an identity of 31% between the two copies. The average molecular weight of all the triose phosphate isomerases is 27 kDa with an identity between them greater than 85%, except for SCO0578.

Another *Streptomyces* gene that showed genetic redundancy was the one coding for glyceraldehyde 3-phosphate dehydrogenase. Glyceraldehyde 3P-dehydrogenase enzyme shows two classes of proteins, some with 331–335 amino acids (approx. 36.1 kDa) and others with a higher molecular weight, the majority of 481 residues (52.9 kDa), except for *S. venezuelae* with 461 amino acids (50.1 kDa). Only *S. albus* has a single small protein and the rest of the microorganisms had two or three, one large and one or two small. *S. hygroscopicus* has four paralogues, three of approximately 335 amino acids and one of 481 residues. Alignment of the amino acid sequences of glyceraldehyde 3P-dehydrogenase showed that the larger proteins (SLAV\_31635, SPRI\_6547, SVEN\_0459, CP970\_08935, SNOUR\_35775, SHJGH\_1800, SCO7040, SGR\_936, and BB341\_01000) had an extended N-terminal region of approximately 126 residues. The highly conserved regions GFGRIGR, ASCTTNA, PRVPV, and WYDNEXG were near or into the NAD-binding and C-terminal domains of the enzyme (**Figure 2**). In the phylogenetic analysis, the sequences are divided into two lineages. The first clade is formed by small-sized proteins, whereas the other included all proteins with large-size copies. Small protein clades are split into two subgroups. One is formed by a single organism with the copy with less divergence, and the other subgroup is divided into two clades, each containing a single homolog of each organism; therefore, the copies diverged to the point of being more related between species than between duplicates of the same species (**Figure 2**).

In all antibiotic-producing *Streptomyces,* the only reaction whose enzyme is encoded by one gene is 2,3-bisphosphoglycerate-dependent phosphoglycerate kinase (EC <IP_ADDRESS>), which catalyzes the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate with the generation of an ATP molecule. All proteins were 403 amino acids long and had high identity with each other. The phosphoglycerate kinase of *S. clavuligerus*, an overproducer of clavulanic acid, was detected in the proteome of this microorganism grown in tryptic soy broth, indicating that its gene was expressed in this culture medium [12].

Phosphosphoglycerate mutase (EC <IP_ADDRESS>) performs the internal transfer of a phosphate group from the C-3 carbon to the C-2 carbon, resulting in the isomerization of 3-phosphoglycerate to 2-phosphoglycerate. Only two genes are involved in this activity in *S. griseus, S. clavuligerus, S. coelicolor*, and *S. noursei*. Most of the proteins are 252–253 amino acids in length, which are very similar (identity greater than 90%), and there are only two proteins with 511 amino acids long, SLNWT\_3230 and SCO6818 also, with 82% identity and 91.6% similarity between them. One of the two *S. noursei* mutases (SNOUR\_07945) is smaller (218 amino acid residues), with 22% identity to 253 amino acid mutases and 92% identity to histidine phosphatases of various *Streptomyces*. Then probably, this mutase is wrongly annotated in its genome.

Enolase (EC <IP_ADDRESS>), also known as phosphopyruvate hydratase, is a metalloenzyme responsible for converting 2-phosphoglycerate to PEP. *S. coelicolor*,

**Figure 2.**

*Partial multiple alignment of glyceraldehyde 3-phosphate dehydrogenase proteins (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

*Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

*S. griseus*, *S. lavendulae*, S. *pristinaspiralis*, and *S. venezuelae* have two enolases, one with 426–428 amino acids and the other with 432–435 residues. The rest have only one enolase, which in most of these *Streptomyces* has 426 amino acids. Between the two proteins of different sizes, there is approximately 56% identity; however, between the small proteins of the different microorganisms, there is an identity greater than 90%, and between the larger ones, there is an identity greater than 83%.

Pyruvate kinase (EC <IP_ADDRESS>) is a key enzyme involved in the last step of glycolysis that catalyzes the transfer of a phosphate group from PEP to ADP, yielding one molecule of PYR and one molecule of ATP. There are two types: type I and type II, and both enzymes show positive cooperative effects concerning PEP. The type I enzyme is activated by fructose 1,6-bisphosphate (F1,6BP) and the type II by AMP [13]. According to the amino acid sequences of PYKF and PYKA enzymes from *E. coli*, the smaller *Streptomyces* PYR kinases are type I, and the others are type II. Most proteins have 474–479 amino acids regardless of whether they are class I or class II and have greater than 60% identity between them.

### **4. TCA cycle**

The TCA cycle is a central metabolic pathway of all aerobic organisms and synthesizes many important precursors and molecules [14] and eight reactions are performed for complete glucose oxidation.

The first reaction is citrate synthase (CS, EC <IP_ADDRESS>), which catalyzes the irreversible conversion of OXA and acetyl-CoA into citrate. The proteins are classified into two types: type I and type II, and are encoded by four genes in eight of the selected antibiotic-producing *Streptomyces*, three in *S. venezuelae*, two in *S. noursei,* and *S. pristinaespiralis*. Their coding genes show the greatest redundancy among all the genes involved in carbon metabolism in most *Streptomyces* species (**Table 2**). Sals\_SLNWT\_1427 (*S. albus*) and ska\_CP970\_28100 (*S. kanamyceticus*) are the largest, with 439 and 451 amino acid residues, respectively. In the amino acid sequence alignment, two groups of CSs are distinguished: small ones ranging from 363 to 395 amino acid residues and large ones ranging from 416 to 451 residues. Small or large types of either type I or II have been found, implying that type does not depend on size. As shown in **Figure 3**, a highly conserved histidine is embedded in the conserved region GPLHGXA. Another conserved amino acid is arginine in the DPR conserved amino acid sequence and aspartic or glutamic acid in the conserved NVD/E. All of these were previously reported as essential residues involved in interactions with the substrate OXA in *Streptomyces* [15]. All CSs have a common ancestor but are separated into two main lineages. One of them is split into two subgroups, which include CS classified as types 1 and 2, like the proteins of *S. lavendulae* (type 1) and *S. pristinaespiralis* (type 2) with approximately 429–433 amino acid residues. The other clade was also split into two subgroups, including proteins with 366 and 390 residues. The CSs of *S. coelicolor* encoded by *sco2736*, *sco4388* (both classified as type 2), and *sco5832* (unclassified) are grouped in one of the main clades and distributed in the two subgroups. The sequence encoded by *sco5831* is grouped into the other principal clade with another seven proteins. The % of identity among the proteins included in each subgroup is 87%–94% and similarity 91%–98%. These differences are mainly observed at the amino terminus. Taking *S. coelicolor* CSs as an example, the identity between the CSs found in each subgroup, was low, between 24.6% and 28.1%, and the similarity between 46% and 49%.

Aconitase (EC <IP_ADDRESS>), isocitrate dehydrogenase (EC <IP_ADDRESS>), the E1 component of 2-oxoglutarate dehydrogenase (EC <IP_ADDRESS>), and malate dehydrogenase (EC <IP_ADDRESS>) are encoded by unique genes in all *Streptomyces,* probably because of the

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

**Table 2.**

*Gene multiplicity in TCA cycle genes.*

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

#### *Actinobacteria - Diversity, Applications and Medical Aspects*

#### **Figure 3.**

*Partial multiple alignment of citrate synthase proteins (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

importance of the reactions they catalyze. The molecular weight of the aconitases of these microorganisms is 97 kDa with an identity between them greater than 92%. Isocitrate dehydrogenases have a molecular weight of 79 kDa in all these *Streptomyces*, except *S. noursei* and *S. pristinaspirales*, which also have another protein (SNOUR\_17135 and SPRI\_3067) that are smaller (45 kDa). The identity between the large proteins and between the small ones is about 85% and 77%, respectively. On the other hand, all MDHs have 329 amino acids with an identity between 87 and 93%.

The 2-oxoglutarate dehydrogenase complex is a central enzyme in aerobic metabolism that catalyzes the oxidative decarboxylation of oxoglutarate, generating

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

NADH [16]. 2-oxoglutarate dehydrogenase is composed of three subunits, E1 (EC <IP_ADDRESS>), E2 (EC <IP_ADDRESS>, dihydrolipoamide succinyltransferase), and E3 (EC <IP_ADDRESS>; dihydrolipoamide dehydrogenase). As previously mentioned, there is a single gene coding for component E1 in all selected antibiotic-producing *Streptomyces*. The E2 component is encoded by one gene, except in *S. coelicolor*, which has three. *S. pristinaespiralis*, *S. albus*, *S. clavuligerus*, *S. coelicolor*, *S. griseus*, and *S. hygroscopicus* genomes have only one gene coding for E3 component, while the remaining five microorganisms have two copies with a low identity (≤ 38%) between them suggesting different origins.

Multiple alignments of the amino acid sequences of the E2 subunit of 2 oxoglutarate dehydrogenase showed close resemblance, with many highly conserved amino acids within the conserved domains, such as the YDHR region, which is part of the 2-oxoacid dehydrogenase acyltransferase catalytic domain. The proteins encoded by *sco7123* and *sco1268* of *S. coelicolor* were smaller than the rest, with sequences of 372 and 417 amino acids, respectively. In contrast, the one encoded by *sco5281* had 1272 residues, similar to the proteins of the rest of the *Streptomyces* studied. This characteristic was reflected in the phylogenetic tree, where the former two proteins were separated from the others in one clade. Large proteins were grouped in the second clade, with SCO5281 being the less related group member (**Figure 4**).

There is an alternative way to perform the synthesis of 2-oxoglutarate via 2 oxoglutarate ferredoxin oxidoreductase (EC <IP_ADDRESS>) formed by two subunits called a and b. *S. avermitilis, S. griseus*, *S. kanamyceticus*, and *S. noursei* have only one copy of this pair of genes, whereas *S. albus* has two genes for subunit b and one for a, and *S. clavuligerus* and *S. noursei* have two for subunit a and one for b. In contrast, *S. coelicolor* has two copies of each, generated by gene duplication because the proteins have 99% identity. *S. lavendulae* also has two copies of each gene, with an identity of 38.4%.

The next reaction is catalyzed by succinyl-CoA synthetase (EC <IP_ADDRESS>), which is also composed of two protein subunits: α and β. *S. albus*, *S. clavuligerus*, *S. griseus*, *S. lavendulae*, *S. noursei*, and *S. venezuelae* have a single copy of the genes that code for each subunit. Two copies are present in the genomes of *S. avermitilis*, *S. coelicolor*, *S. pristinaespiralis, S. hygroscopicus, and S. kanamyceticus*; a subunit is smaller (290–299 residues) than b (376–394), one of the copies being around 376 amino acids and the other larger in all cases. In the first four microorganisms, the genes for the a and b subunits are physically together for both pairs of genes, whereas in the latter, they are separated by five genes in both cases (CP970\_05375/CP970\_5370; CP970\_16770/ CP970\_16765). The identity and similarity between the two proteins subunits a or b in each of the *Streptomyces* range from 57.9% to 78.6% and 74.8% to 86.2%, respectively.

Succinate dehydrogenase (SDH; EC <IP_ADDRESS>, EC <IP_ADDRESS>), which catalyzes the oxidation of succinic acid to generate fumaric acid is a four-subunit multimeric enzyme (iron-sulfur protein, flavoprotein subunit, hydrophobic membrane anchor protein, and cytochrome b-556 subunit). The number of genes that code for each subunit varies, and even the same genome *Streptomyces* can have different number of genes for each subunit. As shown in **Table 2**, the *S. albus* genome contains two genes for the SDH flavoprotein subunit, two for the iron-sulfur protein, one for the SDH hydrophobic membrane anchor protein, and two for the SDH cytochrome b-556 subunit. *S. avermitilis* has three genes for flavoprotein subunit, while *S. coelicolor* has four. On the other hand, all of these microorganisms have one copy of the gene coding the hydrophobic membrane anchor protein and two for the cytochrome b-556 subunit.

The genes that code for iron-sulfur protein have great redundancy, finding three copies in all antibiotic-producing *Streptomyces* except *S. albus*, which has two. In total, 28 proteins were found that have very similar molecular weights, however, according to the identities between them, three groups can be distinguished. The

#### **Figure 4.**

*Multiple alignment of 2-oxoglutarate dehydrogenase component E2 (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

#### **Figure 5.**

*Partial multiple alignment of malate dehydrogenase proteins (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

first includes proteins of 246–249 amino acids and that have an identity between them greater than 88% and is one of the copies in all these microorganisms. In another group are those with 252 amino acids and identities greater than 94%. The last group includes proteins with 256–267 amino acids with identities greater than 85%. All these data indicate that although the subunits have very similar molecular weights, they are actually not so similar, suggesting that they had a common ancestor but that they have diverged a lot over time.

It has been found that there are three different flavoprotein proteins, some with 649–652 amino acids with identities greater than 90%, another group of proteins with 584 amino acids and identities between 91 and 93%, and the last group with 633–667 residues and identities greater than 80%. The flavoproteins SCO7109 and SHJGH\_5964 do not resemble each other or the proteins of the previous groups.

The hydrophobic membrane anchor proteins are smaller than the previous ones, around 17 kDa, and present identities between 77% and 82%, while there are two types of cytochrome b subunit, one of around 223–243 amino acids with lower identities than the previous ones, between 58% and 71%. The second type includes proteins of smaller size and identity between 74% and89%.

The hydration reaction of fumarate to generate malate is catalyzed by fumarate hydratase (EC <IP_ADDRESS>; fumarase). There are two classes of proteins: fumarase classes I and II. Most antibiotic-producing *Streptomyces* have a single gene for fumarase Class I, while only seven of these *Streptomyces* have fumarase class II (**Table 2**). The class I proteins are larger (60.2 kDa) than the class II proteins (50.3 kDa). The identity among class I fumarases and class II is 74% and 75%, respectively.

The final TCA cycle reaction is catalyzed by malate dehydrogenase, which catalyzes the reversible conversion of malate to OXA using NAD<sup>+</sup> or NADP<sup>+</sup> as the coenzyme [17], which is only encoded by a single gene in all microorganisms that produce different antibiotics. The multiple sequence alignment of this enzyme showed a high degree of conservation between them, distributed in two main clades from a common ancestor, which was then subdivided into eight subclades (**Figure 5**). The identity is higher than 90%.

#### **5. PEP-PYR-OXA node**

The PEP-PYR-OXA node is a major branch of central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle [7]. A large variety of enzymes involved in the node have been reported, such as PEP carboxylase (EC <IP_ADDRESS>), pyruvate carboxylase (EC <IP_ADDRESS>), PEP carboxykinase (EC <IP_ADDRESS>), malic enzymes (EC <IP_ADDRESS>), pyruvate kinase (EC <IP_ADDRESS>), and pyruvate phosphate dikinase (EC <IP_ADDRESS>). These enzymes are indispensable for distributing PEP, PYR, and OXA in *Streptomyces*. The enzymes involved in this node vary among microorganisms, and their activity depends on the culture conditions [2, 18].

This anaplerotic pathway does not present multiplicity in any of the genes that encode the enzymes participating in the PEP-PYR-OXA node, except for malic enzymes and pyruvate phosphate dikinase as shown in **Table 3**. PYR carboxylase is an enzyme that is present only in *S. albus*, *S. coelicolor*, *S. hygroscopicus*, and *S. pristinaspiralis*, with only one copy of the gene. The molecular weight is 121 kDa and with 77%–88% identity and 88%–95% similarity among all proteins.

PEP carboxylase, an enzyme that catalyzes the carboxylation of PEP to OXA, is encoded by a single gene and is present in all antibiotic-producing *Streptomyces*. The

**Table 3.** *Gene multiplicity in PEP-PYR-OXA*

 *node.*


**1. Introduction**

Recently, hydrogen has started to be regarded as an alternative ocean fuel source. As a result, studies on hydrogen-fueled fuel cells have been actively conducted in Europe and the United States [1–3].

Fuel cells are known as a technology in which the chemical energy associated with hydrogen molecules is converted to electricity and thermal energy through electrochemical reactions with air. Thanks to the minimization of emissions, the noise in operation and high adaptability of various fuel sources, fuel cells are considered a next-generation technology for clean production [4,5].

In particular, fuel cell-based power plants are expected to reduce greenhouse gas emissions by 30% compared to fossil fuel-based power generation. Considering such benefits, many developed countries have been carried out a number of projects to stimulate the fuel cell application to industries [4–10].

According to a report of the European Maritime Safety Agency (EMSA), since the first project 'US SSFC' was launched in 2000, 24 projects have been made available to facilitate the application of marine fuel cells. Eleven of them selected the Proton Exchange Membrane Fuel Cell (PEMFC) type composed of polymer resin with several advantages: Relatively low operating temperature, about 80 ◦C; shorter time to reach the operating temperature; unnecessary of peripheral devices [11].

On the other hand, to ensure the reliability of the stakes, it requires pure hydrogen which is operated in a very low temperature with the late response time. In order to reduce such drawbacks, expensive catalyst and electrode are applied [12–14].

To obtain pure hydrogen, a separate reforming system is additionally required. That limits the application of PEMFC for the propulsion of medium-large ships. The Zero Emissions Ships (ZEMShip) project has demonstrated the excellent performance of a fuel cell by applying a 96 kW fuel cell to the Motor Vessel (MV) 'Alsterwasser' [15–19].

In 'FellowSHIP' and 'Molten Carbonate Fuel Cells for Waterborne Application (MC-WAP)' projects, molten carbonate fuel cell (MCFC) type was applied to supplement auxiliary power rather than the main propulsion power. In particular, 'FellowSHIP' from 2003 to 2017, applied MCFC fuel cells for MV 'Viking Lady', 6,000 Deadweight Tonnage (DWT) Offshore Support Vessel (OSV), The project results showed excellent performance of the fuel cell in reducing emission levels [8,20–22].

Because MCFCs can operate at high temperatures, low-cost catalysts are available, which simplifying system design and reducing costs. In addition, even with long voyages, this type of fuel cell can utilize natural gas or coal gas as a direct fuel instead of using an external reformer. These advantages may be suitable for application as a major source of power for the ship's fundamental loads. Despite many research and projects for fuel cell applications in the marine industry, attempts to use the MCFC type for medium and large vessels for propulsion are scarce. Given this background, this study was motivated to investigate the suitability of the MCFC for the large vessel propulsion [23–27].

This paper was focused on analyzing the power quality of each power source in synchronization and breakaway. A test bed with a capacity of 180 kW was constructed using 100 kW fuel cells, 30 kW batteries and 50 kW diesel generator.

An actual voyage data for 5,500 TEU container vessel was used to verify the power quality of the fuel cell. Three load scenarios were developed by examining the performance characteristics of the fuel cell, battery and diesel generator systems based on normal navigational conditions. Each developed scenario was applied to the hybrid power sources, and the quality of voltage and frequency was examined during synchronization and breakaway phases.


**5. Conclusions**

In this study, we present a novel AAA-ATPase member gene, *OsAAA-ATPase1*, one of the six AAA-ATPase genes upregulated in *OsSSI2*-kd rice plants [12]. Functional analysis revealed that *OsAAA-ATPase1* is transcriptionally regulated by SA, and plays a positive role in the SA-mediated disease resistance in rice plants. Our findings provide novel insights into SA-regulated defense activation in rice, and the molecular basis of defense activation in *OsSSI2*-kd rice plants.

**Author Contributions:** Conceived and designed the experiments: X.L. and C.-J.J. Performed the experiments and analyzed the data: X.L., H.I., X.T., Y.T., X.X., and C.W. Interpreted and wrote the manuscript, H.I. and C.-J.J. All authors have read and agreed to the manuscript as written.

**Funding:** This work was supported by the National Natural Science Foundation of China (31370306), and grants from Japan's Society for the Promotion of Science (JSPS) KAKENHI to C.J. (19K06065) and to H.I. (17K07678).

**Acknowledgments:** We thank the NARO Genebank for providing the cDNA clone of *OsAAA-ATPase1* (*Os03g0802500*).

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


**1. Introduction**

In order to tackle the development of advanced nuclear technologies, the reliability of passive systems has become an important subject and area under discussion, for their extensive use in new and advanced nuclear power plants, (NEA, 2002), in combination with active safety or operational systems.

Following the IAEA definitions, [1], a passive component does not need any external input or energy to operate and it relies only upon natural physical laws (e.g. gravity, natural convection, conduction, etc.) and/or on inherent characteristics (properties of materials, internally stored energy, etc.) and/or 'intelligent' use of the energy that is inherently available in the system (e.g. decay heat, chemical reactions etc.).

The term "passive" identifies a system which is composed entirely of passive components and structures or a system which uses active components in a very limited way to initiate subsequent passive operation. That is why passive systems are expected to combine among others, the advantages of simplicity, a decrease in the need for human interaction and a reduction or avoidance of external electrical power or signals. These attractions may lead to increased safety and acceptability of nuclear power generation if the detractions can be reduced.

Besides the open feedback on economic competitiveness, special aspects like lack of data on some phenomena, missing operating experience over the wide range of conditions, and driving forces which are smaller - in most cases - than in active safety systems, must be taken into account: the less effective performance as compared to active safety systems has a strong impact on the reliability assessment of passive safety systems.

A categorisation has been developed by the IAEA in [1] distinguishing:

© 2012 Burgazzi, licensee InTech. This is an open access chapter 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. © 2012 Burgazzi, licensee InTech. This is a paper 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.

a. physical barriers and static structures (e.g. pipe wall, concrete building).

This category is characterized by:

Examples of safety features included in this category are physical barriers against the release of fission products, such as nuclear fuel cladding and pressure boundary systems; hardened building structures for the protection of a plant against seismic and or other external events; core cooling systems relying only on heat radiation and/or conduction from nuclear fuel to outer structural parts, with the reactor in hot shutdown; and static components of safety related passive systems (e.g., tubes, pressurizers, accumulators, surge tanks), as well as structural parts (e.g., supports, shields).

Reliability of Passive Systems in Nuclear Power Plants 25

previous categories except that internal intelligence is not available to initiate the process. In these cases an external signal is permitted to trigger the passive process. To recognize this

Examples of safety features included in this category are emergency core cooling and injections systems based on gravity that initiate by battery-powered electric or electropneumatic valves; emergency reactor shutdown systems based on gravity or static pressure

According to this classification, safety systems are classified into the higher categories of passivity when all their components needed for safety are passive. Systems relying on no external power supply but using a dedicated, internal power source (e.g., a battery) to supply an active component are not subject to normal, externally caused failures and are included in the lowest category of passivity. This kind of system has active and passive characteristics at different times, for example, the active opening of a valve initiates

Inclusion of failure modes and reliability estimates of passive components for all systems is recommended in probabilistic safety assessment (PSA)1 studies. Consequently the reliability assessment of passive safety systems, defined as the probability to perform the requested

Notwithstanding that passive systems are credited a higher reliability with respect to active ones, – because of the smaller unavailability due to hardware failure and human error -, there is always a nonzero likelihood of the occurrence of physical phenomena leading to pertinent failure modes, once the system comes into operation. In fact the deviations of the natural forces or physical principles, upon which they rely, from the expected conditions can impair the performance of the system itself. This remark is especially applicable to type B passive systems (i.e. implementing moving working fluids) named thermal-hydraulic passive systems, due to the small engaged driving forces and the thermal-hydraulic

Indeed, while in the case of passive A systems the development of the structural reliability analysis methodology can be carried out with the application of the principles of the probabilistic structural mechanics theory, and operating experience data can be inferred for the reliability assessment of passive C and D components, there is yet no agreed approach as

In fact, such passive safety systems in their designs rely on natural forces, such as gravity or natural convection, to perform their accident prevention and mitigation functions once actuated and started: these driving forces are not generated by external power sources (e.g., pumped systems), as is the case in operating reactor designs. Because the magnitude of the natural forces, which drive the operation of passive systems, is relatively small, counterforces (e.g. friction) can be of comparable magnitude and cannot be ignored as it is generally

1 In the following PSA (Probabilistic Safety Assessment) and PRA (Probabilistic Risk Assessment) are utilized

mission to achieve the generic safety function, becomes an essential step.

departure, this category is referred to as "passive execution/active initiation".

subsequent passive operation by natural convection.

phenomena affecting the system performance.

far as passive B systems are concerned.

indifferently

driven control rods.

b. moving working fluids (e.g. cooling by free convection).

This category is characterized by:

Examples of safety features included in this category are reactor shutdown/emergency cooling systems based on injection of borated water produced by the disturbance of a hydrostatic equilibrium between the pressure boundary and an external water pool; reactor emergency cooling systems based on air or water natural circulation in heat exchangers immersed in water pools (inside containment) to which the decay heat is directly transferred; containment cooling systems based on natural circulation of air flowing around the containment walls, with intake and exhaust through a stack or in tubes covering the inner walls of silos of underground reactors; and fluidic gates between process systems, such as "surge lines" of Pressurized Water Reactors (PWRs).

c. moving mechanical parts (e.g. check valves).

This category is characterized by:

Examples of safety features included in this category are emergency injection systems consisting of accumulators or storage tanks and discharge lines equipped with check valves; overpressure protection and/or emergency cooling devices of pressure boundary systems based on fluid release through relief valves; filtered venting systems of containments activated by rupture disks; and mechanical actuators, such as check valves and spring-loaded relief valves, as well as some trip mechanisms (e.g., temperature, pressure and level actuators).

d. external signals and stored energy (passive execution/active actuation, e.g. scram systems).

This category addresses the intermediary zone between active and passive where the execution of the safety function is made through passive methods as described in the previous categories except that internal intelligence is not available to initiate the process. In these cases an external signal is permitted to trigger the passive process. To recognize this departure, this category is referred to as "passive execution/active initiation".

24 Nuclear Power – Practical Aspects

This category is characterized by:

structural parts (e.g., supports, shields).

This category is characterized by:

b. moving working fluids (e.g. cooling by free convection).

such as "surge lines" of Pressurized Water Reactors (PWRs).

c. moving mechanical parts (e.g. check valves).

This category is characterized by:

systems).

Examples of safety features included in this category are emergency injection systems consisting of accumulators or storage tanks and discharge lines equipped with check valves; overpressure protection and/or emergency cooling devices of pressure boundary systems based on fluid release through relief valves; filtered venting systems of containments activated by rupture disks; and mechanical actuators, such as check valves and spring-loaded relief valves, as well as some trip mechanisms (e.g., temperature, pressure and level actuators).

d. external signals and stored energy (passive execution/active actuation, e.g. scram

This category addresses the intermediary zone between active and passive where the execution of the safety function is made through passive methods as described in the

a. physical barriers and static structures (e.g. pipe wall, concrete building).

Examples of safety features included in this category are physical barriers against the release of fission products, such as nuclear fuel cladding and pressure boundary systems; hardened building structures for the protection of a plant against seismic and or other external events; core cooling systems relying only on heat radiation and/or conduction from nuclear fuel to outer structural parts, with the reactor in hot shutdown; and static components of safety related passive systems (e.g., tubes, pressurizers, accumulators, surge tanks), as well as

Examples of safety features included in this category are reactor shutdown/emergency cooling systems based on injection of borated water produced by the disturbance of a hydrostatic equilibrium between the pressure boundary and an external water pool; reactor emergency cooling systems based on air or water natural circulation in heat exchangers immersed in water pools (inside containment) to which the decay heat is directly transferred; containment cooling systems based on natural circulation of air flowing around the containment walls, with intake and exhaust through a stack or in tubes covering the inner walls of silos of underground reactors; and fluidic gates between process systems,

Examples of safety features included in this category are emergency core cooling and injections systems based on gravity that initiate by battery-powered electric or electropneumatic valves; emergency reactor shutdown systems based on gravity or static pressure driven control rods.

According to this classification, safety systems are classified into the higher categories of passivity when all their components needed for safety are passive. Systems relying on no external power supply but using a dedicated, internal power source (e.g., a battery) to supply an active component are not subject to normal, externally caused failures and are included in the lowest category of passivity. This kind of system has active and passive characteristics at different times, for example, the active opening of a valve initiates subsequent passive operation by natural convection.

Inclusion of failure modes and reliability estimates of passive components for all systems is recommended in probabilistic safety assessment (PSA)1 studies. Consequently the reliability assessment of passive safety systems, defined as the probability to perform the requested mission to achieve the generic safety function, becomes an essential step.

Notwithstanding that passive systems are credited a higher reliability with respect to active ones, – because of the smaller unavailability due to hardware failure and human error -, there is always a nonzero likelihood of the occurrence of physical phenomena leading to pertinent failure modes, once the system comes into operation. In fact the deviations of the natural forces or physical principles, upon which they rely, from the expected conditions can impair the performance of the system itself. This remark is especially applicable to type B passive systems (i.e. implementing moving working fluids) named thermal-hydraulic passive systems, due to the small engaged driving forces and the thermal-hydraulic phenomena affecting the system performance.

Indeed, while in the case of passive A systems the development of the structural reliability analysis methodology can be carried out with the application of the principles of the probabilistic structural mechanics theory, and operating experience data can be inferred for the reliability assessment of passive C and D components, there is yet no agreed approach as far as passive B systems are concerned.

In fact, such passive safety systems in their designs rely on natural forces, such as gravity or natural convection, to perform their accident prevention and mitigation functions once actuated and started: these driving forces are not generated by external power sources (e.g., pumped systems), as is the case in operating reactor designs. Because the magnitude of the natural forces, which drive the operation of passive systems, is relatively small, counterforces (e.g. friction) can be of comparable magnitude and cannot be ignored as it is generally

1 In the following PSA (Probabilistic Safety Assessment) and PRA (Probabilistic Risk Assessment) are utilized indifferently

the case of systems including pumps. Moreover, there are considerable uncertainties associated with factors on which the magnitude of these forces and counter forces depends (e.g. values of heat transfer coefficients and pressure losses). In addition, the magnitude of such natural driving forces depends on specific plant conditions and configurations which could exist at the time a system is called upon to perform its safety function. All these aspects affect the thermal-hydraulic (T-H) performance of the passive system.

Reliability of Passive Systems in Nuclear Power Plants 27

natural circulation systems are described hereafter and namely as regards AP600/AP1000,

It is important to note that the incorporation of systems based on natural circulation to achieve plant safety and economic goals is being extended also to Generation-IV reactor concepts: however due to the early stage of the design - many systems are not yet

Figure 1 presents a schematic that describes the connections of the primary system passive

**2.1. AP600/AP1000 Passive Residual Heat Removal systems (PRHR)**

**Figure 1.** Passive Safety Systems used in the AP600/AP1000 Designs

A Four Stage Automatic Depressurization System (ADS)

An In-containment Refueling Water Storage Tank, (IRWST)

The PRHR implemented in the Westinghouse AP1000 design consists of a C-Tube type heat exchanger in the water-filled In-containment Refuelling Water Storage Tank (IRWST) as

The AP600/AP1000 passive safety systems consist of: A Passive Residual Heat Removal (PRHR) System

Two Core Make-up Tanks (CMTs)

Two Accumulator Tanks (ACC)

A Lower Containment Sump (CS)

Passive Containment Cooling System (PCS)

ESBWR and ABWR designs.

safety systems.

established - they are not explicitly addressed.

Consequently, a lot of efforts have been devoted mostly to the development of consistent approaches and methodologies aimed at the reliability assessment of the T-H passive systems, with reference to the evaluation of the implemented physical principles (gravity, conduction, etc.). For example, the system fault tree in case of passive systems would consist of basic events, representing failure of the physical phenomena and failure of activating devices: the use of thermal-hydraulic analysis related information for modeling the passive systems should be considered in the assessment process.

The efforts conducted so far to deal with the passive safety systems reliability, have raised an amount of open issues to be addressed in a consistent way, in order to endorse the proposed approaches and to add credit to the underlying models and the eventual reliability figures, resulting from their application. In fact the applications of the proposed methodologies are to a large extent dependent upon the assumptions underlying the methods themselves. At the international level, for instance, IAEA recently coordinated a research project, denoted as "*Natural Circulation Phenomena, Modelling and Reliability of Passive Systems"* (2004-2008), [2,3], while another coordinated research project on "*Development of Methodologies for the Assessment of Passive Safety System Performance in Advanced Reactors"* (2008-2011) is currently underway: while focus of the former project has been the natural circulation and related phenomena, the objective of the latter program is to determine a common analysis-and-test method for reliability assessment of passive safety system performance. This chapter provides the insights resulting from the analysis on the technical issues associated with assessing the reliability of passive systems in the context of nuclear safety and probabilistic safety analysis, and a viable path towards the implementation of the research efforts in the related areas is delineated as well. Focus on these issues is very important since it is the major goal of the international research activities (e.g. IAEA) to strive to reach a common consensus about the different proposed approaches. The chapter is organized as follows: after an overview on passive safety systems being implemented in the design of innovative reactors and an introduction on the main components of Probabilistic Safety Assessment approach, at first the current available methodologies are illustrated and compared, the open issues coming out from their analysis are identified and for which one of them the state of the art and the outlook is presented; the relative importance of each of them within the evaluation process is presented as well.


**2. Results**

#### *2.1. Occurrence of Enteric Parasites*

A total of 1093 children aged 3–14 years participated in the present study, of which 807 children were enrolled from 18 schools and resided in 66 neighbourhoods in 10 districts of the Zambézia province. The other 286 children were enrolled from six primary healthcare centres and a hospital clinic and resided in 37 different neighbourhoods in six districts. Overall, *G. duodenalis* was the most prevalent enteric parasite found [41.7%, 95% confidence interval (CI): 38.8–44.7%], followed by *Blastocystis* sp. (14.1%, 95% CI: 12.1–16.3%), and *Cryptosporidium* spp. (1.6%, 95% CI: 0.9–2.5%). The prevalence rates of these pathogens in each participating school and healthcare centre are summarized in Table 1. Estimates did not consider the clustered nature of the data, as this task was thoroughly conducted elsewhere [27].

**Table 1.** Molecular-based prevalence rates of *Giardia duodenalis*, *Cryptosporidium* spp., and *Blastocystis* sp. in the surveyed paediatric population by school or medical centre of origin in the Zambézia province, Mozambique.

#### *2.2. Prevalence and Molecular Characterization of G. duodenalis*

A total of 456 DNA isolates tested positive for *G. duodenalis* by real-time polymerase chain reaction (qPCR, 336 and 120 in asymptomatic and symptomatic children, respectively). Generated cycle threshold (Ct) values had median values of 31.6 (range: 18.0–42.1) in asymptomatic children, and of 31.2 (range: 19.7–41.4) in symptomatic children. Overall, 45.8% (209/456) had qPCR values ≥32, and 39.7% (181/456) had qPCR Ct values <30. Based on previous molecular studies conducted by our research team using this very same method in human populations from other African countries including Mozambique [23,28,29], only DNA isolates with qPCR Ct values <32 (*n* = 247) were assessed for genotyping and sub-genotyping purposes in order to optimise available resources.

Assemblage/sub-assemblage assignment was conducted by direct comparison of the sequencing results obtained at the three loci (*gdh*, *bg*, and *tpi*) investigated. Sequences presenting double peak positions that could not be assigned unequivocally to a given assemblage/sub-assemblage were reported as ambiguous sequences. Out of the 247 DNA isolates investigated, 15.0% (37/247), 10.1% (25/247), and 6.1% (15/247) yielded amplicons at the *gdh*, *bg*, and *tpi* loci, respectively (Table 2). Overall, 17.4% (43/247) were amplified at least at a single locus, whereas multi-locus genotyping data at the three loci were available for 4% (10/247) of them. Most (88.4%, 38/43) of the isolates successfully amplified at any of the three markers assessed had qPCR Ct values <30. Sequence analyses revealed the presence of assemblages A (7.0%, 3/43) and B (88.4%, 38/43). Two additional sequences (4.6%, 2/43) corresponded to mixed A+B infections. No infections caused by host-restricted canine (C, D), feline (F), or ruminant (E) assemblages were detected. All genotyped isolates except one were obtained in asymptomatic children.

**Table 2.** Multilocus genotyping results of the *G. duodenalis*-positive children (*n* = 43) successfully genotyped at any of the three loci investigated in the Zambézia province, Mozambique.

1 Symptomatic child, *bg*: ß-giardin, *gdh*: glutamate dehydrogenase, qPCR: real-time PCR, *tpi*: triose phosphate isomerase.

All three A sequences were assigned to the sub-assemblage AII of the parasite. Out of the 40 B sequences, 12.5% (5/40) were identified as sub-assemblage BIII, 20.0% (8/40) as

sub-assemblage BIV, 52.5% (21/40) as ambiguous BIII/BIV sequences, and the remaining 15.0% (6/40) were only genotyped at the assemblage level.

The diversity, frequency, and main features of the *G. duodenalis* sequences generated at the *gdh* locus are shown in Table S1. Briefly, all four AII sequences were identical to reference sequence L40510. In contrast, a much higher level of genetic diversity was observed within the 34 sequences assigned to assemblage B at this locus. Indeed, the five sequences identified as BIII differed by 1–9 single nucleotide polymorphisms (SNPs) from reference sequence AF069059, most of them associated to heterozygous C/T peaks at positions 99, 147, 150, 309, and 336. The nine sequences unambiguously assigned to BIV differed by 2–8 SNPs from reference sequence L40508. Six of them presented nucleotide substitutions (mainly C↔T transitions) at positions 183, 387, 396, and 423, but not ambiguous positions in the form of double peaks. SNPs present in the three remaining BIV sequences combined mutations and heterozygous positions at different proportions. Remarkably, virtually all ambiguous BIII/BIV sequences different among them and by 5–15 SNPs from reference sequence L40508. Most of these SNPs involved heterozygous C/T (and, to a lesser extent, A/G) peaks. In contrast, SNPs associated to transition C↔T or A↔G mutations were rare or non-existent. Some of these ambiguous BIII/BIV sequences presented clear double peaks at defined positions specific for BIII (e.g., 99, 147, 150, 309, and 336) and BIV (e.g., 183, 387, 396, and 423) sequences, suggesting the occurrence (at an unknown rate) of true BIII+BIV intra-assemblage mixed infections. Several heterozygous positions within BIII and BIII/BIV (particularly the latter) sequences were potentially associated to amino acid change in the polypeptidic chain.

The diversity, frequency, and main features of the 25 *G. duodenalis* sequences generated at the *bg* locus are summarized in Table S2. The only sequence assigned to AII was identical to reference sequence AY072723. The other 24 sequences, all belonging to the assemblage B of *G. duodenalis*, presented a comparatively lower degree of genetic diversity than their counterparts at the *gdh* locus. Of them, four sequences were identical to reference sequence AY072727, whereas the remaining 20 sequences differed from it by 1–5 SNPs. Variations involving transitional C↔T or A↔G mutations and double peaks tended to accumulate at positions 183, 309, 519, and 565 of AY072727; some of them (including a transversion C/A mutation) were involved in amino acid substitutions at the protein level.

The diversity, frequency, and main features of the 15 *G. duodenalis* sequences generated at the *tpi* locus were summarized in Table S3. Out of the three sequences identified as AII, two of them varied by 1–2 SNPs (including a transversion C/G mutation at position 287) from reference sequence U57897. The third AII sequence lacked sufficient quality to accurately determine the presence of potential SNPs. The 10 sequences characterised as BIII differed by 1–8 SNPs from reference sequence AF069561. Six of them included only transitional C↔T or A↔G mutations, whereas the remaining four had several heterozygous positions. Detected SNPs tended to accumulate at positions 34, 108, and 141 of AF069561, some of them involved in amino acid substitutions in the polypeptidic chain. Two isolates corresponded to ambiguous BIII/BIV sequences differing by seven SNPs from reference sequence AF069560. Most of these SNPs were the result of transitional C/T or A/G mutations, one of them (T57C) involving an amino acid chain at the protein level. No transversion mutations were detected within sequences generated at the *tpi* locus.

The evolutionary relationships among the *G. duodenalis* sequences generated at the *gdh* locus in the present study were shown in Figure 1. Sequences of human origin generated by our research team in previous studies conducted in geographical areas of high (Ethiopia, Angola, Brazil, and Iran) and low (Spain) endemicity were also included in the analysis for comparative purposes. Assemblage A sequences grouped together in well-defined clusters with appropriate reference sequences. Although assemblage B sequences also formed a well-supported clade (88% of bootstrap), sub-assemblage BIII and BIV sequences could not be segregated in independent clusters. Phylogenetic trees generated at the *bg* (Figure S1) and *tpi* (Figure S2) loci seem to corroborate this finding.

**Figure 1.** Phylogenetic relationships among *Giardia duodenalis* assemblages and sub-assemblages identified in infected symptomatic and asymptomatic children in the Zambézia province, Mozambique. The analysis was conducted by a neighbor-joining method of a 412-bp fragment (corresponding to position 79–490 of reference sequence L40508) of the *gdh* gene sequence. Genetic distances were calculated using the Kimura two-parameter model. Green filled squares represent sequences generated in the present study. Purple filled dots represent reference sequences. Bootstrap values lower than 50% are not displayed. *Giardia ardeae* was used as outgroup taxon to root the tree.