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",isbn:"978-1-83968-460-9",printIsbn:"978-1-83968-459-3",pdfIsbn:"978-1-83969-232-1",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"babca2dea1c80719111734cc57a21a4c",bookSignature:"Dr. Amin Talei",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10404.jpg",keywords:"Water Budget, Ground Measurement, Satellite Data, Empirical Models, Physical Models, Data-Driven Models, Artificial Neural Network, Neuro-Fuzzy Systems, Genetic Programming, Irrigation Management, Drought, Aquifer Management",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"October 29th 2020",dateEndSecondStepPublish:"November 26th 2020",dateEndThirdStepPublish:"January 25th 2021",dateEndFourthStepPublish:"April 15th 2021",dateEndFifthStepPublish:"June 14th 2021",remainingDaysToSecondStep:"2 months",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"A pioneering researcher in developing hydrological models using adaptive neuro-fuzzy systems, a pioneering researcher in tropical biofiltration systems, appointed head of the Civil Engineering Discipline in Monash University Malaysia.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"335732",title:"Dr.",name:"Amin",middleName:null,surname:"Talei",slug:"amin-talei",fullName:"Amin Talei",profilePictureURL:"https://mts.intechopen.com/storage/users/335732/images/system/335732.jpg",biography:"Associate Professor Amin Talei joined Monash University Malaysia in January 2013 and currently is the head of Civil Engineering discipline. His previous appointment was as researcher in School of Civil & Environmental Engineering of Nanyang Technological University of Singapore where he studied for his PhD during 2008-2011. His research is predominantly focused on hydrological modeling and flood forecasting using artificial intelligence techniques. Most recently, he has been also involved in research projects dealing with sustainable urban water management. To date, he has published over 50 articles in reputable journals and international conference proceedings. He has supervised several PhD and Master students and won the Supervisor of the Year Award in Monash University Malaysia in 2017. He has absorbed over AUD370,000 research funding from industry and international/national funding agencies since 2014 and is a chartered professional engineer of the Engineers Australia.",institutionString:"Monash University Malaysia",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Monash University Malaysia",institutionURL:null,country:{name:"Malaysia"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"10",title:"Earth and Planetary Sciences",slug:"earth-and-planetary-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"297737",firstName:"Mateo",lastName:"Pulko",middleName:null,title:"Mr.",imageUrl:"https://mts.intechopen.com/storage/users/297737/images/8492_n.png",email:"mateo.p@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"46229",title:"Modern Taxonomy for Microbial Diversity",doi:"10.5772/57407",slug:"modern-taxonomy-for-microbial-diversity",body:'Microorganisms are actually composed of very different and taxonomically diverse groups of communities: archaea, bacteria, fungi and viruses. The members of these groups or taxa are distinct in terms of their morphology, physiology and phylogeny and fall into both prokaryotic and eukaryotic domains. They constitute a broad group of life system inhabiting the known ecosystems on earth: terrestrial and marine; including geographical locations considered to be extreme or inimical to life. The latter comprise of such areas as habitats with high salinity, alkalinity, acidity, high and low temperatures, high pressure, and high radiation. Considering the adaptability of microorganisms to grow and survive under varied physico-chemical conditions and their contribution in maintaining the balance in ecosystems, it is pertinent to catalogue their diversity as it exists. The inability to visualize them with the naked eye precludes effective classification. As such, using the available tools, microorganisms are broadly classified into prokaryotes and eukaryotes and subsequently into various taxonomical units depending on the resources available and required.
The sustenance of life on earth depends on maintaining the diversity of microorganisms. Human intervention is resulting in depletion of biodiversity and many hotspots are also fast losing their endemic biodiversity. While specific data is hard to come by, it is likely that loss of macro life forms also results in loss of the associated microbial species: symbionts as well as the rhizosphere-colonizing microbes. The significant contribution made by microorganisms in ecosystem sustainability as well as the industrially important biomolecules obtained from them: antibiotics, anti-cancer drugs, enzymes, biofuel and various other compounds, implies that cataloguing them is imperative. However, a simple and effective microbial identification system is still far off. The available tools for classification and identification of microorganisms rely on a number of different technologies. This chapter provides an overview of taxonomy tools for understanding prokaryotic and eukaryotic microbial diversity. Taxonomy (or biosystematics) consists of three main parts: classification (arrangement of organisms based on similarity), nomenclature (naming of the organisms) and identification (determining whether an organism belongs to the group under which it is classified and named). Modern biosystematics also includes phylogeny as an integral part of the classification process [1].
The diversity of microbial communities varies within habitats as much as between habitats [2]. This variation can even occur within a few millimetres, suggesting that microbial diversity encompasses more than the documented evidence available. Hence, biogeography is gaining importance as a field of study from microbial diversity point of interest. Many reasons have been postulated to explain this phenomenon. Due to the innately small size of the microorganisms, environmental complexity plays a major role in determining diversity. Spatial heterogeneity is likely to lead to the formation of many niches within a habitat [3]. Recent tools like metagenomics aid in biogeography studies by providing information on nucleic acid sequence data, thereby directly identifying microorganisms (see Section 9). Therefore the phylogenetic information can be used to compare microbial diversity profile across habitats [2].
Generally, diversity within a particular location and in a community is called alpha diversity. Beta diversity measures the community composition between two or more locations while gamma diversity applies to a region, across continents and biomes and is larger in size than that used for measuring alpha diversity [4].
The evolutionary relationship of microorganisms is called phylogeny. Understanding phylogenetic profiles of microbes becomes a daunting task because of their small size and the lack of particular indicators that could serve as markers. Some proteins and genes are considered as evolutionary chronometers which measure the evolutionary change [5]. Currently, the 16S rDNA sequence is considered to be most reliable for measuring evolutionary relationships in bacteria and archaea (detailed in Section 7.2.1) and the 18S sequence for fungi (see Section 8). However, it is necessary to choose the correct protein or gene for such studies. Such a gene or protein should have certain features which make it most appropriate for deriving evolutionary relationship. The most important criterion is that it should be present in all members of the target group and be functionally homologous in the organisms. The molecule must contain regions of conserved sequences for comparison purposes. The changes in sequence data must be at a slow enough rate to permit measurement so that it may also reflect evolutionary change for the entire group [5].
In the current system of classification, based on the 16S rDNA sequence, evolutionary relationships form the basis for division and three major domains have been recognized, out of which two comprise of bacteria and archaea (prokaryotes) and the third domain is of eukaryotes [6]. It is important to understand evolution in the context of biodiversity. Evolution leading to new ecotypes/species is achieved in many ways. Some species with quick generation times also undergo mutation frequently leading to novel species or strains [3]. Horizontal gene transfer (HGT), via transformation, transduction or conjugation, also accounts for introduction of genes into distantly related organisms, thereby introducing new traits and also impacting on interaction between species and thereby ecosystem processes [7]. It has also been hypothesized that large population sizes of microbes and their low extinction rates may also play a role in maintaining biodiversity, though measurement of such extinction rates is difficult ([8] and references therein).
The universal phylogenetic tree based on comparative ribosomal RNA sequences (adapted from [6]).
The phylogenetic tree representing all living organisms shows that, evolution of current forms of life occurred from a common ancestor (the universal ancestor), depicted by the root (see Figure 1). Two domains are of prokaryotic systems of life: the archaea and Eubacteria; in contrast to previous systems of classification, wherein, the prokaryotes were confined to a single kingdom. However, it is intriguing to note that, genomic studies have shown the archaea to contain unique gene sequences which are not present in bacteria or eukaryotes. Certain genes are also shared between all the three domains. The genes required for core cellular functions are the ones which are necessary for survival of a cell and could have arisen from the common ancestor.
The divergence of the organisms represents the differences in genetic sequences which could have become fixed in each group as they evolved. It is also postulated that earlier, HGT played a key role in transfer of genes between organisms early in the evolutionary history [6]. It occurs as a response to any change in the environment and provides for better adaptation ([9] and references therein). Subsequently, reproductive isolation could have prevented extensive exchange of genes, though it continues to occur amongst prokaryotes.
The bacteria and archaea have evolved along different lines though both are essentially prokaryotic. The archaea are considered to be the most primitive and are common inhabitants of the so-called extreme habitats (hot springs, deep sea hydrothermal vents, alkaline and acidic habitats). Though the bacteria and archaea share certain common features, the archaea also share similarities with eukaryotes which are further exemplified by the 16S rDNA-based phylogenetic analyses.
An array of diverse definitions have been proposed to describe microbial species. Currently, a polyphasic approach is used to define a microbial species using phenotypic and genotypic properties [1, 10]. Whenever a new taxon is proposed, it is essential that the organism be isolated in pure culture and its characteristic features be tested under standard conditions [11]. Whether an organism constitutes a member of a common species is primarily based on whether its DNA-DNA re-association values are more than 70% and melting temperature (ΔTm) is less than 5ºC, the experiments being performed under standard conditions [12]. All the strains within a species must show similar phenotypes. A designated type strain of a species constitutes the reference specimen for that species [13]. A species description must preferably be based on the characteristics of more than one type strain. To be assigned a different species name, members must show at least one and is governed by the [12]. If the 16S rDNA sequences of organisms are ≤ 98.7% or ≤ 97% identical, they are members of different species. This is considered even in the absence of DNA-DNA hybridization experiments since this level of divergence in 16S rDNA sequences constitutes less than 70% DNA-DNA similarity [14]. Uncultured microbes cannot be assigned to a definite species since their phenotype is not known; however, they can be assigned a ‘Candidatus’ designation provided their 16S rRNA sequence subscribes to the principles of identity with known species [15]. A concept applying to a taxon lower than that of the strain is the ecotype – those microorganisms that occupy an ecological niche and are adapted to the conditions of that niche [16]. It is important to remember here that the nomenclature of a taxon is very important as it serves to maintain effective communication across microbiological disciplines and it is governed by the Bacteriological Code [17, 18].
Various techniques used in polyphasic taxonomy for characterization of prokaryotes.
While the species is accepted as the basic unit of taxonomy (see Section 6), sub-species, strains and ecotypes occupy lower distinctive taxonomic levels for certain groups of organisms and are not mandatory for all. When classifying a new taxon, it is essential to describe phenotypic, genotypic and phylogenetic information as accurately as possible. This constitutes the polyphasic approach of taxonomy [1] and is shown in Figure 2. The phenotypic information comes from the colony characteristics, cell type, cell wall-type, pigmentation patterns, proteins and other chemotaxonomic markers while genotypic features are derived from the nucleic acids (DNA / RNA). Phylogenetic information is obtained from studying sequence similarities of the 16S rRNA or 23 S rRNA genes in case of bacteria and 18S rRNA in case of fungi. Many types of molecules are used for delineating and describing a taxon; some are mandatory (16S rRNA genes, phenotypes, chemotaxonomy) while others are optional (amino-acid sequencing of certain protein products, DNA-DNA hybridization), unless required for appropriate description.
The phenotypic methods are all those that do not include the DNA/RNA sequencing or their typing methods. Study of morphological characteristics and chemotaxonomic profiles is broadly associated with phenotypic characterization.
The phenotypic features are the foundation for description of taxa. The morphological, biochemical and physiological characteristics provide in-depth information on a taxon. The morphology can include the colony characteristics (colour, shape, pigmentation, production of slime etc.). Further, the features of the cell are described as to shape, size, Gram reaction, extracellular material like capsule, presence of endospores, flagella presence and location, motility and inclusion bodies. Light microscopy is generally used to describe the broad cell features; however electron microscopy is recommended for high resolution images [18]. The biochemical and physiological features describe growth of the organism at different ranges of temperature, pH, salinity and atmospheric conditions, growth in presence of anti-microbial agents, production of various enzymes and growth in presence of different sole carbon and nitrogen sources [1]. These tests have to be carried out using standardized procedures to obtain results that are reproducible within and between laboratories [19].
Analyses of huge volumes of phenotypic data to derive meaningful relationships amongst a large number of microorganisms can be carried out using computer programs [20]. This system of analyses is called numerical taxonomy. Giving numerical weightage to each trait is followed by analyses of the data by the computer programs generating data matrices between each pair of isolates according to the degree of similarity. Based on the similarity data, cluster analyses are carried out (based on different algorithms) and dendrograms (‘trees’) are generated showing the overall pattern of similarity/dissimilarity amongst the various organisms being studied. While 16S rDNA sequences have garnered attention in recent times as sole means of bringing out the uniqueness of a species; numerical taxonomy (based on phenotypic traits of a large number of species) compares favourably with that of genotypic data and, indeed, is in alignment with the latter [1].
The peptidoglycan component of cell walls of bacteria does not provide much information except for classifying into Gram-positive, Gram-negative and acid-fast bacterial types. However, those in Gram-positive cells contain different types of peptidoglycan depending on the genus or species [21]. The peptidoglycan structure can be analysed by determining its type (A or B), mode of cross-linking (whether it is directly linked or via interpeptide bridge and with amino acids in the bridge), and the composition of amino acids (especially the diaminoacid) of the side chain [18]. While the mode of cross-linkage can vary within a species and also between strains, the amino acid composition is common to all species within a genus. In archaea, pseudomurein is present where N-acetyl muramic acid is replaced by N-acetyl talosuronic acid [22].
Different types of lipids are present in bacterial cells. Polar lipids are present in the lipid bilayer of the cytoplasmic membrane. The diversity of polar lipids is known to be large and many are yet to be structurally elucidated. While in archaea, polar lipids are of types phospholipids, aminophospholipids, glycolipids and phosphoglycolipids, in bacteria, apart from the ones seen in archaea, there are also lipids derived from amino acids, capnines, sphingolipids (glycol or phosphosphingolipids) and hopanoids [18]. In Gram-negative bacteria, lipopolysaccharides are present in the outer membranes. The type of sugar present and the fatty acid type, the linkage of the fatty acid to the sugar (amide or ester linkage) provide information on characteristic of the cell [23−25]. However, determination of lipopolysaccharides is not routinely used in recent times. The total cellular fatty acids are extracted, esterified and the methyl ester content is analyzed by gas chromatography. Under standard conditions, this provides a reliable estimate of taxonomy up to genus and sometimes species level. The technique has been automated and the Sherlock MIS system (MIDI Inc.) has developed an extensive database. Though incomplete, it still is the most widely used system in recent times [18].
The respiratory isoprenoid quinones are components that occur in cytoplasmic membranes of prokaryotes (archaea as well as bacteria). The naphthoquinones (with sub-types phylloquinone and menaquinone) and benzoquinones (ubiquinones, rhodoquinones and plastoquinones) are the major types of quinones. The variability they depict in their side chains in terms of length (5-15 isoprenoid units known till date), degree and position of saturation are of taxonomic significance and help in characterization to various levels of genus and species [26]. These features generally also mirror the 16S rDNA groupings. Isoprenoid ether-linked side chains are present in members of the archaea. They are of different types such as diethers, hydroxylated diethers, macrocyclic diethers, tetraethers, and polyol derivatives of the tetraether [18]. Non-isoprenoid based-ether-linked lipids are present in bacteria and can be straight chain or with simple branched side chains or with mono-unsaturated derivatives [18].
Other than the principal diagnostic methods described above, other techniques used to lesser levels or for comparison between species or strains comprise of the following:
Whole cell protein analyses, wherein protein is extracted from the cells and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This can help in comparison between related strains ([1] and references therein) and also shows congruency with DNA-DNA hybridization results [27]. However, since the identity of the protein bands is not known, this technique suffers from a drawback that is not associated with fatty acid analysis.
Polyamines are a group of compounds in the cytoplasm that provide stability to the DNA and maintain osmolarity in the cell. They are useful to distinguish above genus level and at species level too [28].
Cytochromes are associated with the cytoplasmic membrane and are involved in respiratory and photosynthetic electron transfer. They are ‘heme’ proteins with a ‘heme’ prosthetic group attached to a protein. These are not used alone in identification since there are limited types of distinct cytochromes [29].
Many advanced analytical techniques such as Pyrolysis Mass Spectrometry, Fourier Transform Infrared spectroscopy and UV Resonance Raman Spectroscopy have been employed to determine chemical composition of bacterial cells, primarily the bioactive metabolites from drug discovery point of view and relate it to characteristics of microbes from which the metabolites are obtained [30, 31]. Pyrolysis Mass Spectrometry is a high-resolution technique, wherein microbial colonies are carefully picked and placed onto a iron-nickel foil, vacuum desiccated, heated rapidly and the pyrolysate bombarded with low-energy electrons. The ionized fragments are separated on the basis of their mass to charge ratio (m/z), detected and amplified by an electron multiplier [30]. Metabolites with m/z ratio of 51-200 constitute degradation products useful for taxonomical discrimination, since those with m/z less than 50 are produced by most biological materials and those with m/z ratio greater than 200 are not useful in discriminating taxons. The multivariate data is further analysed by Principal Components Analysis (PCA) to understand the variance while reducing the dimensionality of the data.
Fourier Transform Infrared spectroscopy (FTIR) is a simple and cost-efficient method and has been applied to identify discriminative features of strains. Most cellular components (fatty acids, proteins, carbohydrates and nucleic acids) can be analysed by this method to reveal strain-specific features [32]. Five IR spectral regions or \'windows\': W1 (3000–2800 cm–1) for fatty acids, W2 (1700–1500 cm–1) for amide I and II bands of proteins and peptides, W3 (1500–1200 cm–1) for a mixed region of fatty acid bending vibrations, proteins, and phosphate-carrying compounds, W4 (1200–900 cm–1) for carbohydrates of cell walls and W5 (900–700 cm–1) which is the \'fingerprint region\' with unique absorbances specific for different taxa [33]. The differences in spectra are resolved using multivariate tools such as cluster analysis, discriminant analysis etc. [34].
UV Resonance Raman Spectroscopy (UVRR) uses a frequency of Raman spectra when biological materials are not subjected to fluorescent background while using IR or visible excitation [35]. Where an IR spectroscopy needs hundreds of cells, Raman spectroscopy does not require this [33, 36]. It can also be used with single cells, as demonstrated by Rösch et al. [37], in combination with a data classification approach [38] and can also provide information about the Gram-type of a bacterium [39] as well as relate to moles G + C content [40].
Modern taxonomy has been influenced by genetic methods and indeed, much of the classification and identification is predicated on specific gene sequences. All the techniques involving DNA or RNA fall under genotypic methods.
The technique, which is very nearly a gold standard for taxonomic purposes today, is sequencing of the 16S rRNA gene of bacteria. The 23S rRNA gene sequence is also considered in many studies but lack of comprehensive databases for comparison is a drawback. Since the 16S rRNA is present in all bacteria, is functionally constant and is composed of conserved and variable regions, it has consistently served as a good taxonomic marker for deriving taxonomic relationships [1]. While it has proved to be the foundation for modern taxonomy, there are certain caveats and it has to be considered along with other techniques for formal identification purposes, especially at the species level. Generally universal sets of primers are used to amplify the 16S rRNA gene product gene (~1.5 kB). Subsequently, the amplified product is sequenced and quality of the sequence checked. The sequence is then aligned against high quality sequence data from curated databases [ARB (www.arb-home.de), RDP (http://rdp.cme.msu.edu/), SILVA (www.arb-silva.de) and LTP (www.arb-silva.de/projects/livingtree/)]. Multiple alignment programs such as CLUSTAL_X, CLUSTAL W, CLUSTAL X2, CLUSTAL W2, MEGA, T-COFFEE, MUSCLE) can also be used with manual editing [18].
As mentioned earlier, it has been shown that < 97% similarity of two 16S rDNA sequences implies a different species ([18] and references therein). This cut-off value is generally considered for ecological studies [41]. For actual taxonomic studies, a 98.5% cut-off value is considered [42]. However, the values should be based on high-quality and almost full-length sequences. When the similarity values are ~95%, other methods must also be included to justify the creation of a new genus. The descriptions must also provide information on the differences between the potentially new and existing genera. Subsequent to alignment, phylogenetic trees or dendrograms have to be constructed to reveal the taxonomic position of an organism. Different treeing algorithms such as maximum-parsimony, maximum-likelihood methods are preferred for evaluation of taxonomic position. Inclusion of type strain or type species is essential.
Determination of moles percent guanosine and cytosine constitutes a classical method of establishment of genomic content. This is now being used along with other genotyping methods to establish taxonomic position of an organism [1]. Within species, the G+C content ranges within 3% and within genera 10% [42]. Overall, the G+C content ranges from 24-76% in bacteria.
This method is an indirect measurement of sequence similarity between genomes. A cut-off value of 70% similarity is considered for establishment of species [1]. However, the method has to be reproducible between laboratories and performed under standardized conditions, which is often a drawback. Hence it is applied only where 16S rRNA gene sequences show similarity values above 98%. There have been reports where 16S rRNA gene sequence has shown 99% similarity and yet DNA-DNA hybridization values have been 60% or less. Hence, this method has to be used with caution and performed under highly standardized conditions.
Earlier, sub-typing was done on the basis of biochemical profile (biotyping), serological profile (serotyping), phase susceptibility (phage typing) or antibiotic susceptibility. But currently DNA-typing methods are preferred due to their reproducibility, ease of performance and high level of discrimination between strains [1]. Genotyping methods such as Restriction Fragment Length Polymorphism (RFLP), Randomly Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Amplified Ribosomal DNA Restriction Analysis (ARDRA), Repetitive Element-Polymerase Chain Reaction (REP-PCR), Ribotyping and Multi Locus Sequence Analyses (MLSA) are some of the newer methods to characterize a taxon.
RFLP was one of the earliest methods to be used and consisted of extraction of whole-genome DNA, restriction digestion using specific restriction enzymes and visualization of the DNA bands using gel electrophoresis. However, complex patterns can be generated, making comparison difficult. AFLP makes use of restriction analyses followed by PCR amplification to select for particular DNA fragments. Two restriction enzymes are used and fragments are amplified which can be genus and species-specific. Thus the method,though tedious, can be used for identification and typing purposes [1]. A PCR-based methodology makes use of random oligonucleotide primers 10 bases in length (RAPD), followed by amplification under specified conditions and analyses of the bands for similarity in size after gel electrophoresis. Ribotyping is another technique in which total genomic DNA is extracted, followed by restriction digestion and separation by electrophoresis [43]. Subsequently, the fragements are hybridized with a radiolabeled ribosomal operon probe (genes for 16S, 23S, tRNA and 5S rRNA) and analysed for presence of bands by autoradiography [44]. A simpler technique is amplifying the 16S rDNA (with or without spacer regions) using universal primers and restriction analysis (ARDRA). It can be used to discriminate at species level ([45]; [1] and references therein). Ribotyping and ARDRA have been shown to produce reproducible and congruent results in Lactobacillus sp. [46]. Consensus sequences complementary to repetitive sequences in the genomes of bacteria are used as primers, PCR-amplified and visualized as distinctive bands (REP-PCR). This method is discriminatory and rapid up to species or intraspecific level but is based on a library-based approach [47]. MLSA focuses on sequencing of housekeeping (6-8 protein-coding) genes and phylogenetic analyses of the same. It is a new method for characterizing a species [48]; however, databases are limited for realising the full-extent of utility of this method. Average Nucleotide Identity (ANI) of all orthologous genes in complete genome sequences has been proposed as a method to define species. Limited studies show that 95% ANI corresponds roughly to 70% DNA-DNA similarity values [49]. However, more datasets are required to implement this method.
Fungi are important from industrial point of view as well as the increasing numbers of pathogens that are arising. Primarily, fungi are classified on the basis of appearance, the structure appearing above ground. The below-ground vegetative structures are difficult to identify [50]. The focus is on the asexual stage. The concept of species is somewhat difficult to interpret in the case of fungi since sexual mating does not occur in all fungi; meiosis occurs only in sexual fungi and even where mating occurs, the product of fusion could either be sterile or fertile. The biological concept of species can therefore be applied to sexual fungi. In the case of asexual fungi, similarities in characteristics provide a system for classification. Modern developments such as analyses of DNA by sequencing have brought about a paradigm shift in fungal taxonomy. The phylogenetic species concept is being favoured over the other definitions, especially where asexual fungi are considered. The evolutionary relationships amongst fungi have not been well-delineated. Traditionally, fungal phylogeny has been classified using morphology (involving primarily the fruiting body), cell wall composition [51], cell ultra-structure [52], cytology [53] and metabolism [54] and even based on the study of fossils [55]. Nomenclature also creates confusion since fungi can exist in sexual and asexual stages and can develop at different times in different substrates and relationship between the two states has to be established first.
Currently, modern developments underscoring the importance of phylogeny in bacterial classification has also been used for fungal taxonomy. Molecular genotyping methods such as restriction analysis of internal transcribed spacer (ITS) region, 18S rDNA and RFLP are being used to classify fungi [50]. However, the datasets are not extensive enough to permit effective identification [56]. Hibbett et al. [57] proposed a phylogenetic-based classification for fungi with 195 taxa. The 5S, 5.8S, 18S and 25S rDNAs are considered for phylogenetic studies. Of these, the 18S rDNA has been more extensively used for filamentous fungi [58]. The D1 and D2 variable regions of 25S rDNA are used for yeasts. Limited datasets are available where both 18S and 25 S rDNAs have been sequenced leading to difficulties in comparison [59]. The 25S rDNA allows for comparison to species level [60] while 18S rDNA has to be nearly completely sequenced to obtain pertinent information. The ITS region is suitable to reveal close relationships [61]. The 5S sequence provides information suitable for order level [62].
Metagenomics has emerged as a promising field of interest, where identification of uncultured microorganisms is attempted. Since 99% of the microbial population is considered to be uncultivable, metagenomics assumes importance [63]. Next generation Sequencing (NGS) has fuelled interest in this field. Classical metagenomics analyses samples by extracting environmental DNA followed by de novo sequencing, or amplification of 16S/18S rDNA using specific primers while functional metagenomics focuses on amplification of genes of interest (generally antibiotics, enzymes etc.) and their cloning into select target microorganisms to produce the metabolite of interest. Roche 454 pyrosequencing and Illumina are the most widely used NGS technologies [4]. DNA bar-coding approach is gaining popularity for assessing microbial diversity [64]. Though only limited datasets (especially for eukaryotic microbes) are currently available, the scenario is improving due to faster and cheaper sequencing methods. Inherent differences in microbial evolution rates, chimeric DNA sequences, artefacts generated during PCR or sequencing and non-universality of primers preclude derivation of a common threshold for taxonomic units [65]. However, bioinformatics handles some of these issues. Sequence quality is checked for series of Ns (nucleotides that are unresolved), errors in primer sequences are checked and verified, and sequences where length varies from the expected length [66] are assessed. Programs have been developed to remove pyrosequencing as well as PCR errors ([4] and references therein). After error-checking and trimming, the sequences are aligned, distance matrices calculated and used for clustering the Operational Taxonomic Units (OTUs) using programs such as MOTHUR [67]. OTUs represented by single sequences (singletons) are also documented and can overestimate diversity. Removal of singletons has not been shown to affect alpha diversity much [4] though more studies are required in this regard. Beta diversity remains conserved without singletons but diversity patterns may change in their presence [68].
The definition of a virus ‘species’ is: "A virus species is a polythetic class of viruses that constitutes a replicating lineage and occupies a particular ecological niche" [69]. A virus isolate can refer to any virus as long as the virus has existed for some time. Viruses are not considered to be either prokaryotes or eukaryotes but have implication from health point of view; hence characterization of viruses has increased considerably. Where earlier, only electron microscopy was used, today sequencing of viral genomes constitutes advancements and the database is increasing. According to International Committee on Taxonomy of Viruses (ICTV), proposals are afoot to accept online descriptions of viral taxa based on taxonomical details such as : dsDNA, ssDNA, rtDNA, rtRNA, dsRNA, ssNRNA, ssPRNA, SAT (Satellites), VIR (Viroids), UN (unassigned).
Zinger et al. state (see Pg. 2 of Ref. [4]): “In its broadest meaning, measuring biodiversity consists of characterizing the number, composition and variation in taxonomic or functional units over a wide range of biological organizations (from genes to communities)”. The taxonomical classification of microorganisms has been difficult due to their small size, short generation times and confounded by genetic exchange between unrelated organisms. These limitations have been largely overcome by modern developments of sequencing technologies and the recognition of rDNA sequences as a cornerstone for identification purposes. Overall, it is important to recognize that microbial diversity is intricately linked to its environment and this correlation has to be established by description of environmental parameters whenever sampling is carried out. It is also important to study the phenotypic characteristics and link them to the observations obtained from genotyping techniques. The link between habitat and diversity then becomes easier to understand for future studies.
The modern metal forming industry has taken complete advantage and benefit offered by the advanced techniques in order to remain in today’s competitive market. The solidification modeling is a phase-change phenomena which is amazingly complicated as well as critical in many areas of science and engineering and also very vital in the field of automotive and aerospace applications. In the field of foundry engineering, when the molten metal is poured into the mold cavity, the metal solidifies and discharges heat into the mold, the metal shrinks due to which an air gap is formed in between the cast and the mold. This air gap acts as an obstruction for the heat flow from the cast to the mold and is to be found as one of the moving boundary conditions to be given as input for the casting simulation software. In the simulation of a solidification of the casting process, many parameters play a significant role responsible for the quality of the cast.
\nThe data base for the properties of commonly used materials such as density, thermal conductivity, specific heat, solidus temperature, liquidus temperature, latent heat release etc., for the simulation of casting parameters need to be maintained by the industries.
\nTo comprehend the heat transfer mechanism we need to know the behavior of solidification. The heat transfer from the liquid hot temperature cast to the mold is a very complex phenomenon and different modes of heat transfer can be observed while solidification in the cast. While heat transfer is predominant the resistance to the heat flow also has different dimensions to this solidification. This resistance mainly depends on liquid cast metal, latent heat release, interface, solidified cast, the type of mold and the ambient conditions. General solidification of an alloy is discussed in the Figure 1 and specific cooling curve for Al6061 is shown in Figure 2.
\nSolidification curve for alloy.
Aluminum alloy (Al6061) solidification curve.
Initially on pouring the liquid metal cast into the mold cavity the whole metal fluid flows and occupies the mold cavity, the liquid metal flowing with the velocity, mixes thoroughly and releases heat to the mold due to the very high temperature difference. Complete thermal contact is observed between the cast and the mold which causes the heat transfer to be purely conduction, where the resistance offered by this liquid metal is negligible since the entire fluid flow is the superheated cast metal. Once the cast metal reaches the liquidus point on cooling, the cast shrinks and releases latent heat and also a number of metal oxides are released which causes an air gap between the cast and the mold. Due to this air gap the heat transfer phenomenon now changes to a complex one where all modes of heat transfer can be observed simultaneously. This air gap is characterized with an Interfacial Heat transfer Coefficient (IHTC) “h” across the metal-mold interface. The rate of heat at the interface is found using the surface heat flux as q (W/m2) and given by the Eq. 1.
\nTc and Tm are the cast and mold surface temperatures at the interface in K or deg. C.
\nThe dynamics of solidification of cast metal, mold temperature and the cast temperature can be clearly understood from the cooling curves shown in Figure 2. Once the molten metal fills the cavity the alloy cast reaches the maximum temperature. Generally the heat transfer analysis starts from this point onwards as the temperature drops from the liquid cast metal to the liquidus temperature (TL), the point at which the solidification begins and this freezing is called liquid cooling. The loss of superheat temperature of the cast metal after pouring is found due to the turbulence in the liquid metal. This rate of cooling is linear and a minimum amount of heat is transferred from the cast to the mold as it is having a complete contact with the mold surface.
\nAs the solidification progresses with time it reaches the liquidus point at the same time where the mold temperature increases significantly to a maximum temperature. Further the solid skin forms on the outer cast surface, the metal shrinks and an air gap starts forming between the metal and the mold. When the cast solidifies further the air gap separates the two surfaces. This is a common phenomenon in most of the alloys. The rate of heat transfer from the cast to the mold is very high as it releases larger quantity of latent heat to the mold and the cast temperature gradually reaches a solidus (TS) temperature of the alloy. The air gap plays a significant role in varying IHTC with various factors influencing solidification.
\nFurther solidification reduces the cast surface temperature, however the inner cast metal shrinks and it further releases the heat to the mold and there is rise in the mold temperature as shown in Figure 2. Thereafter further reduction in the cast temperature after the solidus point (Ts) was found as the third stage of solidification. The air gap size is further increased as the solidification time increase and its effects are felt till the end of solidification. However there is still a temperature difference between the cast and mold for the further heat transfer to continue.
\nOnce the complete air gap is formed between the cast and the mold, the gap will contain almost all kinds of gaseous except air that contradicts the air gap term. The sand mold which is used for the casting application, generates the mold gases which are often high in hydrogen, containing typically 50 percent which fills the air gap. The hydrogen gas thermal conductivity increases the heat transfer by 7 times more as the mold temperature rises to a high temperature of 500°C due to radiation. Therefore it is very essential to know or analyze the interface during the solidification process as it is further discussed in the next section.
\nOn comparing the green sand mold with dry sand mold the green sand mold expand homogeneously and release heat to the surrounding which leads to a lesser resistance for the heat flow whereas dry sand mold offers more resistance than the green sand mold. The high thermal conductivity die mold material has uniform temperature variation and assumes homogeneous expansion.
\nWhile melting the metal in the furnace has a higher specific volume hence it occupies more space by the metal and on pouring it results in the solidification in the mold which increases the complexity of the solidification [1]. After pouring the temperature of the cast reduces and the specific volume also reduces which causes shrinkage in the poured volume as shown in Figure 3. To understand the complex behavior of solidification we need to understand three different stages of shrinkage of metal during the solidification process; it includes liquid shrinkage, liquid- solid shrinkage and solid shrinkage.
\nSpecific volume changes against cast surface temperature.
The superheated metal which is poured in the liquid state has more specific volume than the liquid metal in the cavity [2]. This liquid metal occupies the mold cavity and is in superheated state and comes in complete contact with the mold surface. Here the mode of heat transfer is purely conduction shown in Figure 3. On solidification there is a liquid contraction due to reduction in specific volume, the metal cools further and reaches to a liquidus temperature. This contraction of liquid metal separates cast and mold surface and imitates the air gap formation which is assigned as liquid shrinkage.
\nActually the liquid contraction leads to a solidification which is a complex problem in the casting industry. This requires a proper feeding mechanism to fill the cavity by maintaining high liquid cast temperature while pouring and if not then the partial liquid - solid contraction leads to shrinkage porosity. The specific volume of the solid metal is lesser than the liquid metal. All the solidifications are planned for the directional solidification which refers to the faster cooling rate at which solidification progresses from the cavity metal to the feeder mechanism. The faster cooling rate and the movement of liquid in the solidification is due to the area of the surface which enables the liquid metal to drop its high temperature to solidus temperature. The runner, riser and the gating system is designed in the mold pattern enhances the directional solidification by transferring proper heat flow from the cast to the mold.
\nThe alloys of eutectic type allow lesser solidification shrinkage volume and also have a lower sensitivity to the solidification problems caused by sudden geometry changes. While they involve smaller risers, these can be omitted completely in certain cases by gates placed strategically and because the metal feed avenues stay open longer, it ensures a uniform solidifying process. While eutectic type of solidification is the most simplest, it requires the least reciprocity and can withstand a range of geometries. Directional solidification is more complex; however, when it has an ideally designed geometry, it is highly capable of extremely higher interior unity. Heat transfer is in fact the main process behind the bilaterally symmetrical and mutual state of connectedness in the process of solidification shrinkage and geometrical patterns. The heat transfer during solidification of castings involves three modes of heat transfer, namely radiation, conduction and convection, the rate of heat transfer is still dependent on the geometry of the casting as discussed later in the interfacial heat transfer coefficient section.
\nThe final stages of shrinkage in the solid state which can cause a separate series of problems. As cooling progresses, and the casting attempts to reduce its size in consequence, it is rarely free to contract as it wishes. This stage of solidification is usually complex either by the types of mold, or by the other casting parts like the runner and riser that have already solidified and cooled as the air gap formed. The air gap formed is mainly due to the various factors like metal oxide formation, coefficient of thermal expansion, latent heat released, evaporation moisture in the case of sand mold, interfacial gap, mode of heat transfer etc. this type of solidification shrinkage is also called as pattern shrinkage.
\nThese factors are the major causes for the heat to flow from the cast to the mold and it is found that it majorly affects the solidification and in turn affects the quality of the cast product. The amount of solid metal stretches like plastic casting, makes the solidification again into a complex problem. This shrinkage behavior leads to difficulty in predicting the size of the pattern since the degree to which the pattern is made oversize (the ‘contraction allowance’ or ‘patternmaker’s allowance’) is not easy to quantify. This shrinkage also causes hot tearing or cracking of the casting which lead to more localized problems.
\nIn general, liquids contract on freezing because of the rearrangement of atoms from a rather open ‘random close-packed’ arrangement to a regular crystalline array of significantly denser packing. The densest solids are those that have cubic close packed (face-centred-cubic, fcc, and hexagonal close-packed, hcp) symmetry. Thus the greatest values for contraction on solidification are seen for these metals.
\nThe heat transfer characteristics during casting are governed by IHTC. The molten metal is poured into the cavity it first enters the mold due to the fluidity of the metal, it occupies the cavity and ensures complete contact between the metal and the mold. In the early stage of solidification, the fluidity of the molten metal conformance and contact between the cast and mold surfaces is good. At this early stage of solidification due to the nucleation of the metal, higher initial surface heat flux is reached. Further the solid skin forms and then spreads to cover the entire casting surface. As the solidified layer forms with sufficient strength, simultaneously air gap forms and as a consequence the contact between the casting and the mold are reduced. This leads to the sudden drop in the heat flux and the solid skin forms on the outer cast surface [3]. The cast liquid - solid shrinks/contracts away from the mold surface. This further releases heat and it is absorbed by the mold surface and in turn increases the temperature of the mold as it expands. The mode of heat transfer is not only due to conduction at this stage because the heat from the metal to the mold takes place across the interface region but also due to other modes of heat transfer convection and radiation. The air gap varies for the different cast metals and depends on their factors of the release of metal oxides, hydrogen gases and material properties of the cast and mold, geometry etc.
\nFurther the third stage of solidification is identified between the liquidus to solidus temperature of the cast as the fall in the casting surface temperature is suddenly halted, due to the release of latent heat. After the complete solid skin formation on the cast the heat transfer further diminishes and gap size increases and the mode for heat transfer is assumed to be conduction of heat through the gaseous phase in the interface using the air gap method. This air gap size is measured as x by assuming the expansion to be homogeneous, and the interfacial heat transfer coefficient is estimated as h = k/x: where k is thermal conductivity of the air (W/mK) as shown in Figure 4. This concept of conduction as a mode of heat transfer in IHTC is reported by Kai- Ho and Robert D Pelhke, [4]. There are many factors that influence the IHTC and practically the IHTC becomes highly unpredictable if all the factors are not taken into account while designing. The various factors listed by the authors Lewis and Ransing, [5] and Guo Zhi-Peng et al. [6], that affect the interfacial heat during solidification is listed below.
\nSchematic representation of IHTC during solidification of casting.
\n
Die coating thickness: The initial high peak value of IHTC is reduced with an increase of die coating thickness. While pouring the metal at the liquid stage the effect of die coating behaves as a weaker influence at the interface as the air gap formed.
Insulating pads, chills, etc.: The IHTC has different behaviors with insulating pads and chills. It is obvious that always the insulating material reduces the IHTC and the chills increases the IHTC.
Geometry of Casting: The area of contact with the mold and the directional solidification will have higher IHTC.
Pouring temperature: Higher values of superheat will increase the initial value of IHTC.
Surface roughness: Higher initial value of IHTC for the better contact when the surfaces are smooth.
Alloy composition: Higher initial value established for an alloy with a larger freezing range.
Latent heat: Cast from superheat temperature to liquidus temperature ensures sharp slope in IHTC due to the evolution of latent heat.
Metallostatic pressure: During the pouring of molten metal into the cavity rises the metallostatic pressure, this is also responsible for higher IHTC at the initial stage.
Mold temperature: During initial stage higher IHTC due to the higher mold temperature and smaller temperature difference for higher peak heat flux.
Die Coating thickness: Increase of die coating thickness decreases the IHTC. While pouring the metal at the liquid stage the effect of die coating behaves as a weaker influence at the interface as the air gap formed.
Mold materials
Type of castings
As it is pointed out by many researchers the gap size mainly depends on the gas that is formed in the interface. The rate of solidification of castings made in a sand mold is generally controlled by the rate at which heat can be absorbed by the mold. In fact, compared to many other casting processes, the sand mold acts as an excellent insulator, keeping the casting warm. However, of course, ceramic investment and plaster molds are even more insulating, avoiding premature cooling of the metal, and aiding fluidity to give the excellent ability to fill thin sections for which these casting processes are renowned. It is regrettable that the extremely slow cooling can contribute to rather poorer mechanical properties.
\nExtensive literature reviews have been made, in order to determine the interfacial heat transfer behavior during the solidification of casting at the metal-mold interfaces, since the 1970’s. The boundary conditions as a surface heat flux and mold surface temperature established at the metal mold interface were used to determine the precise interfacial heat transfer coefficient value by using many mathematical methods described in the literature. The most common approaches can be distinguished here as follows for the determination of IHTC at the metal-mold interface including surface heat flux and mold surface temperature:
Air gap measurement technique
Pure Analytical approach
Semi-analytical method
Numerical Methods
The following section explains the detailed procedure of these methods listed above.
\nThis method calculates the IHTC based on entrapped gas properties present at the interface. The thermal conductivity of the air between the cast mold interface and the distance of air gap measured as x with the LVDT [7]. The formula used for IHTC calculation is, h = k/x, W/m2K. The mode of heat transfer assumed in this method is conduction at the interface, but the other modes of heat transfer are also practically possible as we have discussed in the above section. Hence this method is not widely accepted by the researchers.
\nIn this approach, experimental cooling curves were obtained at certain locations of the cast surface and on the mold to estimate the IHTC. The IHTC is calculated based on measured cast temperature, estimated mold surface temperature and estimated mold surface heat flux. Generally solidification heat transfer problems as shown in Figure 5 were categorized as
Direct Heat Conduction Problem (DHCP)
Indirect Heat Conduction Problem (IHCP)
Schematic diagram for DHCP and IHCP conditions.
In the DHCP the boundary conditions were known at the metal mold interface (which is a moving boundary problem and is difficult to acquire the parameters at the interface) and the effects were determined, mathematically it is known as a well posed problem. But in solidification of casting, knowing the boundary condition is very difficult because of its high transient nature, moving boundary problem, high temperature region, combination of all modes of heat transfer, etc., at the interface. So the inverse heat conduction problem is used to approach the problem. In order to calculate the boundary condition at the interface as a surface heat flux and surface temperature of the mold, experiments were carried out to determine temperatures in the mold to get the input data. This leads to a method of adoption of an ill-posed problem or the inverse heat conduction problem (IHCP) [8]. This ill-posed nature makes IHCP conduct experimentation to determine the boundary conditions at the interface before it has to be solved from the available data rather than using a DHCP approach.
\nThe interfacial heat transfer coefficient at the cast mold interface can be calculated based on Eq. (1), requiring the transient surface heat flux. Cast and mold surface temperatures are measured using thermocouples during solidification regardless of its uncertainty in the physical measurements. The pure analytical or other methods mentioned above are unable to determine the surface heat flux at the interface. This leads to the numerical approaches and their formulation of inverse heat conduction problem (IHCP) at the interface to determine the boundary conditions. The boundary conditions at the interface are explored or determined by the IHCP. This has been studied by various techniques like FDM, FEM, FVM and CV methods. One of the common and mostly used method is mainly based on the function minimization technique based on the numerically calculated and measured data [6].
\nWhere, F(h) is the minimization function, Ti, Yi are calculated and measured transient temperatures at the same locations, i= 0 to N, nodal point. The errors in the temperature measurement may also lead the IHCP into ill-posed. This problem leads the researchers to propose many techniques to solve for IHCP to determine boundary conditions at the interface with the measured temperature histories.
Polynomial extrapolation method: The temperature at the interface was deduced by extrapolating any one of the polynomial curve fitting techniques. This method needed many measurements inside the cast and mold surfaces. This mathematical tool failed to minimize measurement errors.
Regularization method: In order to minimize the error from the measurement obtained a sensitivity analysis can be carried out using the Tikhonov regularization theory. This was used to regularize some function to relate the measured data and this was improving the accuracy and stability of the results obtained. This method could achieve an excellent solution and could be applied to any complex geometry, but the computation takes a very long time.
Boundary element method and Laplace transform: the unknown temperature were transformed into equations as well as written as matrix format. This could be easily solved and written into a computer program. But it has some restrictions. It was an effective method to solve a simple linear problem. But the measured temperature data always has more noise (disturbances) in the data, this could fluctuate the result obtained as heat flux.
Beck’s function specification with finite difference method (implicit & explicit): It was another minimizing error technique used based on heat flux, where sum of squares of assumed and calculated data are used into the function. This method could be used for linear or nonlinear problems. Also, it has long computation time and also could achieve an accurate solution with efficient computation.
Control volume method: This method works, based on energy balance applied over a control volume drawn on each nodal point. The next one is the governing equation for the transient heat conduction written as a partial transient heat conduction equation changed into an ordinary differential transient equation. This involves both energy and mass conservation on each node, leads to a complex formulation equation containing up to 4th order, which may be difficult to program using computer languages, and can only be applied to simple geometrical shapes and one dimension.
A sample of a rectangular geometry with an aluminum (Al6061) cast volume of 45 cm3 was solidified and the IHTC was calculated as shown below in Figure 6. Here the IHTC curve was calculated using the control volume method and it shows a gradual increase. Various characteristics of the IHTC and the heat transfer can be discussed [9].
\nIHTC variation for the rectangular aluminum casting with sand mold.
The behavior of the sample rectangular cast was considered as it summarizes most of the heat transfer modes in solidification of the cast. On pouring the IHTC was found to be 370 W/m2 K at 90 s, the higher initial surface heat flux was due to a perfect thermal contact. As further solidification starts, vaporization takes place in the sand mold because of the moisture content, presence of hydrogen release along with metal oxides across the interface and the reduction of specific volume of metal creates an air gap and decreases the value of IHTC rapidly to a minimum value of 163 W/m2 K at 130 s. The shrinkage of metal causes release of latent heat and rise in the IHTC, then heat transfer reduces once the solid skin is formed [10]. Again the inner metal leaks and flows out from the solid skin to outside and gets cooled which again releases latent heat and so IHTC increases and decreases. Continuous rise and fall of the IHTC shows peak formation, which is shown till the end of solidification. The fourth peak value of 1718 W/m2 K at 600 s and further again at 720 s the IHTC reached the highest peak value of 1918 W/m2 K. The vapor pressure developed in the sand mold is due to the escape of moisture content to the ambient, which is sufficient to allow the heat to flow from the solidifying metal to sand mold hence the sharp rise in IHTC is observed in the final stage of solidification. Not only vapor pressure but also huge temperature differences causes high heat flows. Due to the thermal resistance induced, as the metal solidifies and contracts, a fall in the IHTC is vividly observed.
\nThe materials that change phase during solidification to room temperature can be much more complicated. The heat transfer in the solidification is a complicated phenomenon as shown in the above sections. Understanding the heat transfer characteristics while solidification will help to link the various developments in the micro structure of the materials and the dislocations present. When solidification is complete the strength of the material can be assessed and the formation of the grains in the material can be directed by control of the temperature and heat flow on solidification.
\nThe IHTC of a sample of Al6061 is thoroughly explained to comprehend the various modes of heat transfer while solidification is taking place. Proper cooling helps to govern the solidification and as the temperature is sufficiently low the strains of dislocations will not be sufficiently mobile to migrate into low energy positions, forming low-angle boundaries. Thus the alloy will become sufficiently strong to retain any further strain as elastic strain. Once the metal solidifies properly the structure of the alloy will no longer be affected during further cooling. Hence a complete idea of IHTC at all the times of solidification is the best option to minimize the errors and maximize the strength.
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\\n\\nThe Author shall respect confidentiality during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Author and Co-Authors are confidential and are intended only for the recipients. The contents of any communication may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\\n\\nAUTHOR'S WARRANTY
\\n\\nThe Author and Co-Authors confirm and warrant that the Work does not and will not breach any applicable law or the rights of any third party and, specifically, that the Work contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy.
\\n\\nThe Author and Co-Authors confirm that: (i) the Work is their original work and is not copied wholly or substantially from any other work or material or any other source; (ii) the Work has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) Authors and any applicable Co-Authors are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) Authors and any applicable Co-Authors have not assigned, and will not during the term of this Publication Agreement purport to assign, any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\\n\\nThe Author and Co-Authors also confirm and warrant that: (i) he/she has the power to enter into this Publication Agreement on his or her own behalf and on behalf of each Co-Author; and (ii) has the necessary rights and/or title in and to the Work to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licences in this Publication Agreement. If the Work was prepared jointly by the Author and Co-Authors, the Author confirms that: (i) all Co-Authors agree to the submission, license and publication of the Work on the terms of this Publication Agreement; and (ii) the Author has the authority to enter into this biding Publication Agreement on behalf of each Co-Author. The Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each Co-Author.
\\n\\nThe Author agrees to indemnify IntechOpen harmless against all liabilities, costs, expenses, damages and losses, as well as all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of, or in connection with, any breach of the agreed confirmations and warranties. This indemnity shall not apply in a situation in which a claim results from IntechOpen's negligence or willful misconduct.
\\n\\nNothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\\n\\nTERMINATION
\\n\\nIntechOpen has the right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Author and/or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Author and/or any Co-Author (being a private individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Author and/or any Co-Author (as a corporate entity) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for, or enters into, any compromise or arrangement with any of its creditors.
\\n\\nIn the event of termination, IntechOpen will notify the Author of the decision in writing.
\\n\\nIntechOpen’s DUTIES AND RIGHTS
\\n\\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen, at its discretion, agrees to publish the Work attributing it to the Author and Co-Authors.
\\n\\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen agrees to provide publishing services which include: managing editing (editorial and publishing process coordination, Author assistance); publishing software technology; language copyediting; typesetting; online publishing; hosting and web management; and abstracting and indexing services.
\\n\\nIntechOpen agrees to offer free online access to readers and use reasonable efforts to promote the Publication to relevant audiences.
\\n\\nIntechOpen is granted the authority to enforce the rights from this Publication Agreement on behalf of the Author and Co-Authors against third parties, for example in cases of plagiarism or copyright infringements. In respect of any such infringement or suspected infringement of the copyright in the Work, IntechOpen shall have absolute discretion in addressing any such infringement that is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\\n\\nIntechOpen has the right to include/use the Author and Co-Authors names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Work and has the right to contact the Author and Co-Authors until the Work is publicly available on any platform owned and/or operated by IntechOpen.
\\n\\nMISCELLANEOUS
\\n\\nFurther Assurance: The Author shall ensure that any relevant third party, including any Co-Author, shall execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\\n\\nThird Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\\n\\nEntire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by, or on behalf of, the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (known as the "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of any fraudulent pre-contract misrepresentation or concealment.
\\n\\nWaiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\\n\\nVariation: No variation of this Publication Agreement shall have effect unless it is in writing and signed by the parties, or their duly authorized representatives.
\\n\\nSeverance: If any provision, or part-provision, of this Publication Agreement is, or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted. Any modification to, or deletion of, a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\\n\\nNo partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Author or any Co-Author, nor authorize any party to make or enter into any commitments for, or on behalf of, any other party.
\\n\\nGoverning law: This Publication Agreement and any dispute or claim, including non-contractual disputes or claims arising out of, or in connection with it, or its subject matter or formation, shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of, or in connection with, this Publication Agreement, including any non-contractual disputes or claims.
\\n\\nPolicy last updated: 2018-09-11
\\n"}]'},components:[{type:"htmlEditorComponent",content:'When submitting a manuscript, the Author is required to accept the Terms and Conditions set out in our Publication Agreement – Monographs/Compacts as follows:
\n\nCORRESPONDING AUTHOR'S GRANT OF RIGHTS
\n\nSubject to the following Article, the Author grants to IntechOpen, during the full term of copyright, and any extensions or renewals of that term, the following:
\n\nThe foregoing licenses shall survive the expiry or termination of this Publication Agreement for any reason.
\n\nThe Author, on his or her own behalf and on behalf of any of the Co-Authors, reserves the following rights in the Work but agrees not to exercise them in such a way as to adversely affect IntechOpen's ability to utilize the full benefit of this Publication Agreement: (i) reprographic rights worldwide, other than those which subsist in the typographical arrangement of the Work as published by IntechOpen; and (ii) public lending rights arising under the Public Lending Right Act 1979, as amended from time to time, and any similar rights arising in any part of the world.
\n\nThe Author, and any Co-Author, confirms that they are, and will remain, a member of any applicable licensing and collecting society and any successor to that body responsible for administering royalties for the reprographic reproduction of copyright works.
\n\nSubject to the license granted above, copyright in the Work and all versions of it created during IntechOpen's editing process, including all published versions, is retained by the Author and any Co-Authors.
\n\nSubject to the license granted above, the Author and Co-Authors retain patent, trademark and other intellectual property rights to the Work.
\n\nAll rights granted to IntechOpen in this Article are assignable, sublicensable or otherwise transferrable to third parties without the specific approval of the Author or Co-Authors.
\n\nThe Author, on his/her own behalf and on behalf of the Co-Authors, will not assert any rights under the Copyright, Designs and Patents Act 1988 to object to derogatory treatment of the Work as a consequence of IntechOpen's changes to the Work arising from the translation of it, corrections and edits for house style, removal of problematic material and other reasonable edits as determined by IntechOpen.
\n\nAUTHOR'S DUTIES
\n\nWhen distributing or re-publishing the Work, the Author agrees to credit the Monograph/Compacts as the source of first publication, as well as IntechOpen. The Author guarantees that Co-Authors will also credit the Monograph/Compacts as the source of first publication, as well as IntechOpen, when they are distributing or re-publishing the Work.
\n\nThe Author agrees to:
\n\nThe Author will be held responsible for the payment of the agreed Open Access Publishing Fee before the completion of the project (Monograph/Compacts publication).
\n\nAll payments shall be due 30 days from the date of issue of the invoice. The Author or whoever is paying on behalf of the Author and Co-Authors will bear all banking and similar charges incurred.
\n\nThe Author shall obtain in writing all consents necessary for the reproduction of any material in which a third-party right exists, including quotations, photographs and illustrations, in all editions of the Work worldwide for the full term of the above licenses, and shall provide to IntechOpen, at its request, the original copies of such consents for inspection or the photocopies of such consents.
\n\nThe Author shall obtain written informed consent for publication from those who might recognize themselves or be identified by others, for example from case reports or photographs.
\n\nThe Author shall respect confidentiality during and after the termination of this Agreement. The information contained in all correspondence and documents as part of the publishing activity between IntechOpen and the Author and Co-Authors are confidential and are intended only for the recipients. The contents of any communication may not be disclosed publicly and are not intended for unauthorized use or distribution. Any use, disclosure, copying, or distribution is prohibited and may be unlawful.
\n\nAUTHOR'S WARRANTY
\n\nThe Author and Co-Authors confirm and warrant that the Work does not and will not breach any applicable law or the rights of any third party and, specifically, that the Work contains no matter that is defamatory or that infringes any literary or proprietary rights, intellectual property rights, or any rights of privacy.
\n\nThe Author and Co-Authors confirm that: (i) the Work is their original work and is not copied wholly or substantially from any other work or material or any other source; (ii) the Work has not been formally published in any other peer-reviewed journal or in a book or edited collection, and is not under consideration for any such publication; (iii) Authors and any applicable Co-Authors are qualifying persons under section 154 of the Copyright, Designs and Patents Act 1988; (iv) Authors and any applicable Co-Authors have not assigned, and will not during the term of this Publication Agreement purport to assign, any of the rights granted to IntechOpen under this Publication Agreement; and (v) the rights granted by this Publication Agreement are free from any security interest, option, mortgage, charge or lien.
\n\nThe Author and Co-Authors also confirm and warrant that: (i) he/she has the power to enter into this Publication Agreement on his or her own behalf and on behalf of each Co-Author; and (ii) has the necessary rights and/or title in and to the Work to grant IntechOpen, on behalf of themselves and any Co-Author, the rights and licences in this Publication Agreement. If the Work was prepared jointly by the Author and Co-Authors, the Author confirms that: (i) all Co-Authors agree to the submission, license and publication of the Work on the terms of this Publication Agreement; and (ii) the Author has the authority to enter into this biding Publication Agreement on behalf of each Co-Author. The Author shall: (i) ensure each Co-Author complies with all relevant provisions of this Publication Agreement, including those relating to confidentiality, performance and standards, as if a party to this Publication Agreement; and (ii) remain primarily liable for all acts and/or omissions of each Co-Author.
\n\nThe Author agrees to indemnify IntechOpen harmless against all liabilities, costs, expenses, damages and losses, as well as all reasonable legal costs and expenses suffered or incurred by IntechOpen arising out of, or in connection with, any breach of the agreed confirmations and warranties. This indemnity shall not apply in a situation in which a claim results from IntechOpen's negligence or willful misconduct.
\n\nNothing in this Publication Agreement shall have the effect of excluding or limiting any liability for death or personal injury caused by negligence or any other liability that cannot be excluded or limited by applicable law.
\n\nTERMINATION
\n\nIntechOpen has the right to terminate this Publication Agreement for quality, program, technical or other reasons with immediate effect, including without limitation (i) if the Author and/or any Co-Author commits a material breach of this Publication Agreement; (ii) if the Author and/or any Co-Author (being a private individual) is the subject of a bankruptcy petition, application or order; or (iii) if the Author and/or any Co-Author (as a corporate entity) commences negotiations with all or any class of its creditors with a view to rescheduling any of its debts, or makes a proposal for, or enters into, any compromise or arrangement with any of its creditors.
\n\nIn the event of termination, IntechOpen will notify the Author of the decision in writing.
\n\nIntechOpen’s DUTIES AND RIGHTS
\n\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen, at its discretion, agrees to publish the Work attributing it to the Author and Co-Authors.
\n\nUnless prevented from doing so by events beyond its reasonable control, IntechOpen agrees to provide publishing services which include: managing editing (editorial and publishing process coordination, Author assistance); publishing software technology; language copyediting; typesetting; online publishing; hosting and web management; and abstracting and indexing services.
\n\nIntechOpen agrees to offer free online access to readers and use reasonable efforts to promote the Publication to relevant audiences.
\n\nIntechOpen is granted the authority to enforce the rights from this Publication Agreement on behalf of the Author and Co-Authors against third parties, for example in cases of plagiarism or copyright infringements. In respect of any such infringement or suspected infringement of the copyright in the Work, IntechOpen shall have absolute discretion in addressing any such infringement that is likely to affect IntechOpen's rights under this Publication Agreement, including issuing and conducting proceedings against the suspected infringer.
\n\nIntechOpen has the right to include/use the Author and Co-Authors names and likeness in connection with scientific dissemination, retrieval, archiving, web hosting and promotion and marketing of the Work and has the right to contact the Author and Co-Authors until the Work is publicly available on any platform owned and/or operated by IntechOpen.
\n\nMISCELLANEOUS
\n\nFurther Assurance: The Author shall ensure that any relevant third party, including any Co-Author, shall execute and deliver whatever further documents or deeds and perform such acts as IntechOpen reasonably requires from time to time for the purpose of giving IntechOpen the full benefit of the provisions of this Publication Agreement.
\n\nThird Party Rights: A person who is not a party to this Publication Agreement may not enforce any of its provisions under the Contracts (Rights of Third Parties) Act 1999.
\n\nEntire Agreement: This Publication Agreement constitutes the entire agreement between the parties in relation to its subject matter. It replaces all prior agreements, draft agreements, arrangements, collateral warranties, collateral contracts, statements, assurances, representations and undertakings of any nature made by, or on behalf of, the parties, whether oral or written, in relation to that subject matter. Each party acknowledges that in entering into this Publication Agreement it has not relied upon any oral or written statements, collateral or other warranties, assurances, representations or undertakings which were made by or on behalf of the other party in relation to the subject matter of this Publication Agreement at any time before its signature (known as the "Pre-Contractual Statements"), other than those which are set out in this Publication Agreement. Each party hereby waives all rights and remedies which might otherwise be available to it in relation to such Pre-Contractual Statements. Nothing in this clause shall exclude or restrict the liability of either party arising out of any fraudulent pre-contract misrepresentation or concealment.
\n\nWaiver: No failure or delay by a party to exercise any right or remedy provided under this Publication Agreement or by law shall constitute a waiver of that or any other right or remedy, nor shall it preclude or restrict the further exercise of that or any other right or remedy. No single or partial exercise of such right or remedy shall preclude or restrict the further exercise of that or any other right or remedy.
\n\nVariation: No variation of this Publication Agreement shall have effect unless it is in writing and signed by the parties, or their duly authorized representatives.
\n\nSeverance: If any provision, or part-provision, of this Publication Agreement is, or becomes invalid, illegal or unenforceable, it shall be deemed modified to the minimum extent necessary to make it valid, legal and enforceable. If such modification is not possible, the relevant provision or part-provision shall be deemed deleted. Any modification to, or deletion of, a provision or part-provision under this clause shall not affect the validity and enforceability of the rest of this Publication Agreement.
\n\nNo partnership: Nothing in this Publication Agreement is intended to, or shall be deemed to, establish or create any partnership or joint venture or the relationship of principal and agent or employer and employee between IntechOpen and the Author or any Co-Author, nor authorize any party to make or enter into any commitments for, or on behalf of, any other party.
\n\nGoverning law: This Publication Agreement and any dispute or claim, including non-contractual disputes or claims arising out of, or in connection with it, or its subject matter or formation, shall be governed by and construed in accordance with the law of England and Wales. The parties submit to the exclusive jurisdiction of the English courts to settle any dispute or claim arising out of, or in connection with, this Publication Agreement, including any non-contractual disputes or claims.
\n\nPolicy last updated: 2018-09-11
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