\r\n\tIn particular, this book presents topics related to Audio Signal Processing based on the different perspectives of the following: pattern recognition on audio, audio processing, forensic audio, digital filtering, and frequency analysis, and digital signal processing chip for audio, although other topics can be included, too. The most innovative advances on Audio Signal Processing will be included in this book, in order to show the reader, the new researched and developed approaches.
\r\n
\r\n\tSpecific cases of voice applications are welcome, where the Voice over IP (VoIP), internet of things (IoT), deep learning (DL) approaches, etc., are very useful including the recent technologies applied on voice and audio.
",isbn:"978-1-83962-876-4",printIsbn:"978-1-83962-875-7",pdfIsbn:"978-1-83962-877-1",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,hash:"95a662956526e566e5885e68c1d500ed",bookSignature:"Dr. Carlos M. Manuel Travieso-Gonzalez",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8213.jpg",keywords:"Pattern Recognition, Audio Identification, Audio Processing Algorithm, Audio Enhancer, Human Voice Patterns, Text to Speech, Forensic Audio Enhancement, Audio Evidence, Filtering Audio, Wavelet Analysis, Microprocessor for Audio, DSP for Audio",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"October 24th 2019",dateEndSecondStepPublish:"November 14th 2019",dateEndThirdStepPublish:"January 13th 2020",dateEndFourthStepPublish:"April 2nd 2020",dateEndFifthStepPublish:"June 1st 2020",remainingDaysToSecondStep:"a month",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,editors:[{id:"27170",title:"Dr.",name:"Carlos M.",middleName:"Manuel",surname:"Travieso-Gonzalez",slug:"carlos-m.-travieso-gonzalez",fullName:"Carlos M. Travieso-Gonzalez",profilePictureURL:"https://mts.intechopen.com/storage/users/27170/images/system/27170.jpeg",biography:"Dr. Carlos M. Travieso-González received his M.Sc. degree in 1997 in Telecommunication Engineering at the Polytechnic University of Catalonia (UPC), Spain; and his Ph.D. degree in 2002 at the University of Las Palmas de Gran Canaria (ULPGC-Spain). He is a Full Professor and the Head of the Signals and Communications Department at ULPGC a. He is teaching in ULPGC from 2001 on signal processing and learning theory subjects and he has been a supervisor on 8 Ph.D. Thesis (9 more in the process), and 130 Master Thesis. His research lines are biometrics, biomedical signals and images, data mining, classification system, signal and image processing, machine learning, and environmental intelligence. Dr. Travieso-González has contributed research in more than 50 International and Spanish Research Projects, some of them as the head researcher, and is the co-author of 4 books, co-editor of 24 Proceedings Books, Guest Editor for 8 JCR-ISI international journals and up to 24 book chapters. He has over 430 papers published in international journals and conferences (70 of them indexed on JCR – ISI - Web of Science). Dr. Travieso-González has also published 7 patents on Spanish Patent and Trademark Office. He has been a reviewer in different indexed international journals (<70) and conferences (<200) since 2001. Dr. Carlos M. Travieso-González is the Associate Editor on Computational Intelligence and Neuroscience journal and Entropy, and evaluator on European Projects (H2020), ANECA (Spain), DAAD (Germany), MRC (UK), ANR (France) and other institutions. He is a member of IASTED Technical Committee on Image Processing from 2007 and a member of IASTED Technical Committee on Artificial Intelligence and Expert Systems from 2011. Dr. Carlos M. Travieso-González is also the founder of The IEEE IWOBI conference series and President of its Steering Committee, the founder of The InnoEducaTIC conference series and the founder of The APPIS conference series. He was the Vice-Dean from 2004 to 2010 in Higher Technical School of Telecommunication Engineers in ULPGC; and the Vice-Dean of Graduate and Postgraduate Studies from March 2013 to November 2017.",institutionString:"University of Las Palmas de Gran Canaria",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"6",totalChapterViews:"0",totalEditedBooks:"2",institution:{name:"University of Las Palmas de Gran Canaria",institutionURL:null,country:{name:"Spain"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"24",title:"Technology",slug:"technology"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"192910",firstName:"Romina",lastName:"Skomersic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/192910/images/4743_n.jpg",email:"romina.s@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. 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\n\t\t\t
1. Introduction
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Synthetic approaches are widely employed in the emerging research field of systems and synthetic biology, to learn the living organisms in a physical and systematic manner, such as, cellular dynamics and network interactions. Synthetic gene circuits potentially offer the insights into nature’s underlying design principles (Hasty et al, 2002), and genetic reconstructions will give better understanding of naturally occurring functions (Sprinzak and Elowitz, 2005). Technical improvements in synthetic biology will provide not only engineering novelty for applications in biotechnology (McDaniel and Weiss, 2005) but also the fundamental understanding of living systems.
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It is well-known that a library of the parts comprised in the gene circuits, which can be found in MIT Parts Registry (http://parts.mit.edu/), provides a variety for genetic reconstruction. As well, a new born organization (http://biobricks.org/) provides a platform (BioBrickTM parts) for scientists and engineers to work together. Current pioneer studies provided the successful examples of synthetic circuits working in the living cells, such as, the mutual inhibitory circuits functionally constructed in bacterial cells (Gardner et al, 2000), and with newly introduced biological functions (Kashiwagi et al, 2006). However, the reported cases generally do not include the vast majority of many failures. After defining a conceptual design as specifying how individual components are connected to accomplish the desired function, the next step is constructing the well-designed foreign circuit in living cells. So far, the strategies for construction of synthetic gene circuits are more of an art form than a well-established engineering discipline, mostly, in a “Plug and Play” manner (Haseltine and Arnold, 2007).
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Carriers (vector) used for genetic construction are commonly limited in the plasmid, due to the advantageous of its efficiency and easy manipulation. Successful constructions have been reported to mimic a toggle switch in bacterial cells (Gardner et al, 2000), to build a synthetic predator-prey ecosystem (Balagadde et al, 2008), to address the dynamical property of positive feedback system (Maeda and Sano, 2006), to study the behaviour of the synthetic circuit under complex conditions: unregulated, repressed, activated, simultaneously repressed and activated (Guido et al, 2006). However, noise due to the copy number variation in plasmids is inevitable. As know, copy number variation is an important and widespread component within and between cell populations. For example, CNV can cause statistically significant changes in concentrations of RNA associated with growth rate changes in bacteria (Klappenbach et al, 2000; Stevenson and Schmidt, 2004); as well as, small-scale copy number variation can cause a dramatic, nonlinear change in gene expression from the theoretical study on various genetic modules (network motifs) (Mileyko et al, 2008). Thus, low-copy plasmids are utilized for generation of cellular function in the studies of demonstrating that negative auto-regulation speeds the response times of transcription networks (Rosenfeld et al, 2002), identifying heuristic rules for programming gene expression with combinatorial promoters (Cox et al, 2007), studying the biological networks and produce diverse phenotypes (Guet et al, 2002), etc. As well, combination of low-copy plasmid and genome has been applied to analyze the multistablity in lactose operon in bacterial (Ozbudak et al, 2004), to evaluate the fluctuation in gene regulation at the single cell level (Rosenfeld et al, 2005), and to study noise propagation (Pedraza and van Oudenaarden, 2005), and so on. Nevertheless, neither controlling the copy number of plasmid in a living cell nor keeping a constant copy number of plasmid in a growing cell population is easy.
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Difficulties in synthetic approaches of genetic constructions are faced, in particular, as the fact that a stable construction is essential for steady phenotypic quantification. Practical methodology is required for the stable maintenance of the synthetic gene circuits in growing cells. As the genome is the most stable genetic circuit in living cells, insertion synthetic circuit into the genome will promise a best solution. Short fragment genome recombination of a reporter gene is widely applied, particularly, such as, the accurate prediction of the behaviour of gene circuits from component properties (Rosenfeld et al, 2007), and the study on intrinsic and extrinsic noise in a single cell level (Elowitz et al, 2002). It is becoming aware of the importance of genome integration of the synthetic gene networks.
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Though the single copy of genome is the best choice for carrying the synthetic circuit stable along with the cell division and propagation, building a complex synthetic circuit, commonly comprised of a few genetic parts, into genome is not an easy job due to the flowing reasons. Inducing these parts into the genome one by one is time consuming, and the frequently repeated genomic construction process can potentially result in unexpected mutagenesis or stress-induced genomic recombination. The modified method introduced here reduces the frequency of recombination, and provides a time-saving approach for efficient synthetic construction on the bacterial genome. The availability of long insertions allows the easy artificial reconstruction of complicated networks on the genome. The examples of synthetic circuits constructing in Escherichia coli cells using the refined methods are described in detail. An assortment of synthetic circuits integrated into the genome working as design principles are shown. The switch-like response of the synthetic circuit sensitive to nutritional conditions is specially presented. Constructing synthetic gene circuits integrated in bacterial genome is to form a stable built-in artificial structure, and provides a powerful tool for the studies not only on the field of synthetic and systems biology based on bacteria but also on the applications potential for genetic engineering to achieve metabolic reconstruction.
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2. Methodology: Genome-integration of foreign DNA sequences
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As the classic methods for genome recombination, a number of general allele replacement methods have been used to inactivate bacterial chromosomal genes (Dabert and Smith, 1997; Kato et al, 1998; Link et al, 1997; Posfai et al, 1999). These methods all require creating the gene disruption on a suitable plasmid before recombining it onto the chromosome, leading to its complexity in the methodology. A relatively simple method was developed by Wanner’s group, a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted genes (Datsenko and Wanner, 2000). The procedure is based on the Red system that promotes a greatly enhanced rate of recombination over that exhibited by recBC, sbcB, or recD mutants when using linear DNA.
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Elegant applications of Wanner’s method have been reported, such as, the construction of single-gene knock-out mutants (Baba et al, 2006), construction of targeted single copy of lac fusions (Ellermeier et al, 2002), produce insertion alleles for about 2,000 genes systematic mutagenesis of Escherichia coli genome (Kang et al, 2004). Because of the limitation on the insertion length, the optimization on transformation procedure was performed to produce recombinant prophages carrying antibiotic resistance genes (Serra-Moreno et al, 2006). Wanner’s method is very efficient on deletion mutation, even for quite long genome segments, whereas, insertion is limited within 2-3 Kbs technically. The requirement on constructing complicated networks is facing to the technical problem on the length limitation.
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The methodology of genetic construction was recently published as the research article on a new protocol for more efficient integration of larger genetic circuits into the Escherichia coli chromosome. Complex synthetic circuits are commonly comprised of a few genetic parts. Inducing these parts into the genome one by one is time consuming, and the frequently repeated genomic construction process can potentially result in unexpected mutagenesis or stress-induced genomic recombination. The refined procedure introduced here shows the availability of the efficient artificial reconstruction of complex networks on the Escherichia coli genome, and provides a powerful tool for complex studies and analysis in synthetic and systems biology. Comparison between the genome integrated and the plasmid incorporated genes, reduced cell-to-cell variation was clearly observed in genome format. The method demonstrated that the integrated circuits show more stable gene expression than those on plasmids and so we feel this technique is an essential one for microbiologists to use.
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2.1. Refined method
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The method has been modified including medium, temperature, transformation, and selection, as described elsewhere (Ying et al, 2010). The synthetic sequences need to be wholly constructed on a plasmid in advance. Following PCR amplification and purification of the linear target sequence, transformation (electroporation) for genome replacement is performed, to introduce it into competent cells. To distinguish genomic recombinants from the original plasmid carriers, the target synthetic sequence encodes a different antibiotic resistant gene from the original plasmid. False transformants (i.e., transformed colony) carrying the plasmid grow on both antibiotic plates; genomic recombinants grow only on the plate carrying the antibiotic whose resistant gene is encoded in the circuit, but not the one encoded in the plasmid. Dual antibiotics selection for positive transformants reduces the labour and cost of large-scale screening, and uncovers a high ratio of positive candidates on the colony PCR check. The steps of the refined method are described as follows, along with the schematic illustration of the process (Figure 1).
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Figure 1.
Scheme of homologous recombination. The numbering steps are corresponding to the listed procedure of the refined method. Modified from the original paper (Ying et al, 2010).
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Construction of the synthetic sequence on a plasmid (often containing an AmpR gene).
PCR amplification of the target foreign DNA sequence, with the homogenous region corresponding to the recombination site.
Clean-up (buffer exchange or gel extraction) using commercial kits.
Digestion by the enzyme DpnI at 37˚C for 2 h to remove the trace amount of the original plasmid.
Clean-up and condensation of the target sequence. Any commercial kit is convenient.
Transformation to the host strain containing the plasmid of pKD46, encoding the recombinase. Electroporation is crucial.
Culturing in the rich medium (SOC) with 1 mM of arabinose, at 37˚C for 2 h. Quiet incubation often increases the efficiency of transformation.
Plating for antibiotic selection, incubation overnight at 37˚C. Once using a slow growth strain, the additional incubation time is required.
Strike the single colonies onto two plates, each with a different antibiotic, and incubate overnight at 37˚C.
Selection based on the difference of the clones between the two plates: positive candidates exhibited fast growth on the GeneR (the antibiotics resistant gene different from AmpR) plate, and slow or no growth on the AmpR plate. This dual antibiotics screening on the plates promoted the final positive selection by colony PCR.
Colony PCR for final confirmation. This step is essential to make sure that no unexpected recombination occurred in genome, particularly repeated homologous recombination have been performed.
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2.2. High efficiency of recombination
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Synthetic DNA sequences of various lengths (1 ― 10 Kbs) have been inserted into the different sites on genome, such as, intC, argG, glnA, leuB, ilvE, hisC and galK. Comparatively short insertions result in accurate genome replacement. In contrast, longer insertions generally lead to fewer transformants and a worse outcome (i.e., fewer positive colonies); nevertheless, usually there are still sufficient transformants for further selection (Table 1). Genome location (gene site) dependent efficiency of homologous recombination was noticed (unpublished data). The site of galK always gave the best score of successful recombination, regardless of the length of inserted sequences. The efficiency of successful recombination, based on the positive ratio of the colony PCR, depended on the insertion length. Previous studies provide myriad examples of short DNA fragment recombinations (Elowitz et al, 2002), but none of long ones. The ability of this methodology to successfully recombine a foreign DNA fragment of 9 to 10 Kbs indicates that it is possible to construct a complex synthetic gene circuit of a considerable length onto the bacterial genome.
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Table 1.
Recombination efficiency. Genes A, B, C, etc. represent an assortment of genes incorporated into the synthetic circuits. GeneR indicates the antibiotic resistance gene. 1Number of clones grown on an agar plate plated with 300 μL of SOC transformation mixture. 2Ratio of the number of positive clones to the total number of clones applied in the colony PCR test, for final confirmation. Modified from the original paper (Ying et al, 2010).
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In addition, the homologous sequences of various lengths have been evaluated for the transformation efficiency at the identical genome location. In general, a 100-bp overlap, which can be easily generated by PCR amplification, could enable reasonably high genome recombination efficiency. Thought longer homologous sequences are supposed to give higher recombination efficiency, our test showed that length dependency was only present when the homologous sequence ranged from 50 to 150 bps, as an overlap of 300 to 500 bps decreased the number of transformants (Ying et al, 2010). We assume that the accessibility of the secondary structure, caused by the complex conformation of genome, for annealing possibly plays a role in successful recombination. According to the reported studies, genome integration has been generally carried out for 1 or 2 genes for each recombination step. If a relatively large fragment comprising 4 to 8 genes, four cycles of genome recombination procedure are needed. The reduced repetition of homologous recombination procedure of this refined protocol allows long DNA segments exchange at once. It greatly saves time and labour, and is supposed to contribute to the microbiological engineering.
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2.3. Reduced cell-to-cell variation
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Copy number variation in genetic materials can cause large variation within and between cell populations. As reported, it caused significant changes in RNA concentrations associated with growth rate changes (Klappenbach et al, 2000; Stevenson et al, 2004), as well as dramatic differences in gene expression compared to theory in various genetic modules (network motifs) (Mileyko et al, 2008). It is why combination of low-copy plasmids and genomes is currently applied in the studies on fluctuations in living cells (Ozbudak et al, 2004; Rosenfeld et al, 2005).
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The relation between the copy number of the plasmid and the cell-to-cell variation in gene expression has been evaluated by an assortment of experiments using the flow cytometry, as previously reported (Ito et al, 2009). The results showed that the copy number of the plasmid was correlated to the protein abundance, which indicated that the variable in DNA copy number contributed to the cell-to-cell variation in gene expression. The copy number issue is important to be considered, when discussing the property of the target gene and its expression fluctuation. Genomic recombination seems to be the best choice for the studies on biological noise and phenotypic fluctuation, as the genome is a stable carrier of the constant copy number.
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The comparison between the plasmid and the genome clearly verified this statement. Identical synthetic sequence, comprising a reporter gene gfp (green fluorescence protein, GFP) and an antibiotic resistant gene kan\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t, has been transformed into the host Escherichia coli cells, either in a low-copy plasmid (~ 20 copies per cell) or onto the genome. Once both cell types were induced to display similar green fluorescent intensities (i.e., the same averaged concentration of GFP), the variance of the cellular GFP concentration obtained from plasmid carriers was much larger than that from genomic carriers (Ying et al, 2010). It demonstrates that the variety of gene expression levels among the cells carrying the synthetic sequence in plasmid format is much larger than that in the genomic format. Using a very low-copy plasmid (< 5 copies per cell) reduced the cell-to-cell variation but still showed larger variance than the genome format (unpublished data). It greatly suggests that it is noise in the gene copy number that increases the variation in a genetically identical population. In addition, less heterogeneous in the transformant phenotypes and longer generations were often observed in genome-integrated carriers than those of plasmid insertion (personal communication). Taken together, construction of a synthetic gene circuit on the genome will greatly improve the stability of the genetic structure itself and reduce the biological noise from copy number variation.
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3. Applications: Synthetic gene circuits work as design principles
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An applicable protocol for solid and efficient genome recombination is described above. An assortment of well-designed synthetic circuits built-in Escherichia coli cells are going to be introduced as successful applications. Precise reconstruction of synthetic gene circuits to mimic gene expression and regulation is generally employed in a plasmid with a chromosomal reporter gene (Blake et al). Using the modified method, we successful integrated a variety of gene circuits into the bacterial genome. First of all, a relatively long genetic loop of mutual inhibitory structure displayed a strong hysteric expression pattern was successfully constructed, which was assumed to be resulted from the decreased fluctuation due to the genome recombination. Applying the mutual inhibitory design, a bistable dual function genetic switch was built in the genome. Selective expression of the genome integrated genetic switch sensitive to the environmental transition was clearly observed, which suggested that the synthetic circuits could show physiological activity in living cells and play an important role in population adaptation.
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3.1. Mutual inhibitory construct behaved as design principles
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Mutual inhibitory structure has been successfully constructed in bacterial cells on the plasmids (Gardner et al, 2000; Kashiwagi et al, 2006), and the pretty work of this structure in genome format was firstly reported using the refined method (Ying et al, 2010). As shown in Figure 2A, the mutual inhibitory structure, which was integrated into the genome and showed the expected features, was finally acquired using the method described here. The Escherichia coli DH1 cells were used as hosts for the construct. To prevent disturbance of the expression from the promoter Plac by the endogenous level of LacI protein, the native lacI gene and the related genes lacY and lacZ were deleted from the genome. The reporter genes, gfp and rfp, were employed as two different visible phenotypes and were used for quantitative evaluation of the protein levels of the two repressor proteins, TetR and LacI. The two antibiotic resistant genes were used as selection markers during genetic construction. According to the design principle, when the two expression units (i.e., gfp-lacI-kan\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t and rfp-tetR-cat\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t), which are regulated by the promoters Plac and Ptet, respectively, are expressed, they will inhibit each other, and the cells will show a “red” or “green” phenotype. The gfp-lacI-kan\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t unit is highly induced (“green” phenotype) when the chemical inducer, doxycycline (Dox), is added. Similarly, the rfp-tetR-cat\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t unit is greatly induced (“red” phenotype) by isopropyl β-D-1-thiogalactopyranoside (IPTG).
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The engineered Escherichia coli cells carrying this mutual inhibitory structure (Figure 2A) displayed either green or red fluorescent intensity (Figure 2B), representing the two discrete phenotypes of highly induced expression of either unit. As known, the positive feedback structure could accelerate the expression of any of the two expression units occasionally showing slightly higher expression level, while the mutual inhibitory structure would suppress expression of the other unit, leading to a fixation effect of the expressed unit (Gardner et al, 2000; Kashiwagi et al, 2006; Ozbudak et al, 2004). The discrete expression of the two units verified the bistable nature, involved in the mutual inhibitory structure, as designed features.
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Figure 2.
\n\t\t\t\t\t\t\tEscherichia coli cells carrying the mutual inhibitory circuit. A. Genetic construction of the mutual inhibitory structure. B. Immerged image of the Escherichia coli cells observed under fluorescent microscope. Cells carrying the synthetic circuit as illustrated in A were grown in the minimal medium with induced conditions.
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Following preliminary incubation in a doxycycline-free or doxycycline-supplemented (40 nM) medium, Escherichia coli cells carrying the mutual inhibitory structure were cultured in various concentrations of doxycycline (i.e., 0, 5, 10, 15, 20, 30 and 40 nM) to induce the expression of gfp-lacI-kan\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t, the “green” unit. Due to the different initial states, that is, the “green” or “red” induced expression level, the gfp expression levels varied even under the same condition, for instance, in the presence of 5 nM doxycycline, cells with high expression of the “red” unit at preincubation (doxycycline-free condition) showed an induced expression of rfp after incubation (Figure 3, upper panel), while those with high expression of “green” unit in preincubation (doxycycline-supplemented condition) showed an induced expression of gfp after incubation (Figure 3, bottom panel). The hysteresis in gene expression was clearly observed in the engineered Escherichia coli cells, under the inductions of 5 ― 15 nM doxycycline, and kept for several days undergoing serial transfer, equal to approximate 30 to 50 generations. It indicated that the applicable synthetic approach of building genome integrated gene circuits successfully presenting the designed principles.
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Figure 3.
Memory effect of the mutual inhibitory structure. Cells populations growing under the different induced conditions were measured using flow cytometry (FC500, Beckman). Each dot spot represents a single cell. Every 10 000 cells were collected and shown in the density maps here. GFP FI and RFP FI represent the green and red fluorescence intensity, respectively.
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The success of the synthetic design was due to the easy genomic construction method. Construction of a simple toggle switch on a plasmid and transformation into bacterial cells was previously reported (Gardner et al, 2000). A genomic version of the similar toggle switch is introduced here. It could be steadily maintained along with propagation and cell division. Note that, to optimize this structure (i.e., for strong hysteresis of gene expression), the “green” unit (i.e., gfp-lacI-kan\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t) was fixed and the regulatory region of the “red” one (i.e., rfp-tetR-cat\n\t\t\t\t\t\n\t\t\t\t\t\tR\n\t\t\t\t\t) was flexible, by using various operators or promoters. As differences in the sequence of promoters and the number of operators could strongly influence the binding affinity of the repressor proteins, regardless of the identical promoter, the operator sequences have been adjusted to amend the expression level of the repressors. The promoters and operators have been optimized for the enhanced binding affinity with the repressors, strong hysteresis (memory effect) was clearly observed in this construction. Owing to the greatly reduced copy number of the genome integrated synthetic construct, the changes in binding affinity of operator and repressor were clearly observed. The final construct of the satisfying promoter and operator sequences was decided by means of the “Plug and Play” strategy (Haseltine et al, 2007). Thus, optimization of the genetic construct in genome becomes practical, as the modification of the method reduces the steps and increases the efficiency of genome recombination.
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3.2. Synthetic switch sensitive to environmental transitions
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An assortment of synthetic circuits of varied genetic designs has been successfully constructed into the Escherichia coli genome (Kashiwagi et al, 2009; Ying et al, 2010). Experimental investigation demonstrated that these synthetic circuits were functional in living cells and could survive cells from starvation (Tsuru et al, 2011; Shimizu et al, in revision). Among these constructs, a relatively complex circuit of mutual inhibitory structure (Matsumoto et al, 2011) is introduced here.
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3.2.1. Design principles
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As described, bistability can be easily introduced into the genetic design of the synthetic circuit to produce two discrete stable states, “red” and “green”, as shown in Figure 2B. Once the biological functions, for instance, which are crucial for cell growth, are introduced to the synthetic circuit, the two stable states will represent two phenotypes of physiological activities. For instance, two additional genes, geneA and geneB, can be inserted into the two expression units, “red” and “green”, as shown in Figure 4. The geneA and geneB encode the proteins (e.g., enzymes) that catalyze the biological reactions promoting the two independent physiological pathways (Figure 4, Function A and Function B), respectively. Switching between the two expression units is supposed to have selective activation of the two pathways, resulting in growth recovery of cell population and/or causing improved production of target proteins.
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Figure 4.
Synthetic circuit designed for physiological functions. Dual functions are introduced in the mutual inhibitory structure. Induced expression of geneA and geneB represent the activation of Function A and Function B (e.g., amino acid biosynthesis), respectively.
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Figure 5.
Discrete phenotypes designed for the synthetic circuit. Induced expression of either “red” or “green” unit is supposed to be decided by the growing environment, either condition A (e.g., leucine-free medium) or condition B (e.g., histidine-free medium). Selectively induced expression of the two units is shown in red or green.
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Such synthetic gene circuit was finally designed as described elsewhere (Matsumoto et al, 2011) and constructed as follows (Figure 4 and Figure 5). The two expression units, “red” and “green”, representing a dual-function synthetic switch, were built in the Escherichia coli genome by homologous recombination as described in 2.1. The “red” unit contained three genes, rfp, tetR and leuB, encoding a red fluorescent protein (RFP), a repressor protein (i.e., TetR) for blocking expression of the “green” unit (the promoter Ptet) and an enzyme contributing to leucine biosynthesis, respectively. The “green” unit consisted of three genes, gfp, lacI and hisC, encoding a green fluorescent protein (GFP), a repressor protein (i.e., LacI) inhibiting expression of the “red” unit (the promoter Plac) and an enzyme involved in histidine biosynthesis, respectively. That is, the geneA and geneB were replaced by leuB and hisC, and leucine and histidine biosynthesis represented the Function A and B, respectively (Figure 4). By the way, to oblige the cells to use the genes, hisC and leuB, within the synthetic switch, the native regulation of Leu and His operons was disturbed by removing leuB and hisC from their native chromosomal locations. Thus, the expression of leuB and hisC only inside the “red” and “green” units were reported by the red and green fluorescence, respectively. Thus, a synthetic switch based on a mutual inhibitory structure showing two discrete physiological states, the induced leucine (red) and histidine (green) productions, was constructed as designed (details in Matsumoto et al, 2011).
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Rewiring of the stress-stringent genes (i.e., leuB and hisC) to the synthetic circuit allows us not only to investigate the unknown survival strategy in living systems but also to search the possibility of metabolism reconstitution. As genome recombination promises a stable genetic carrier, this synthetic dual-function circuit can be applied to mimic cellular behaviour. According to the design principle, the Escherichia coli cells carrying this synthetic switch are able to show two different phenotypes, “red” and “green”, representing the induced expression of leuB and hisC, and related to two physiological functions, i.e., leucine and histidine biosynthesis, respectively (Figure 5). Bistability, resulting from the mutual inhibitory structure, was assumed to confer the “memory effect” on the cells carrying this structure, as shown in Figure 3, where the same promoter and repressor cassette is used. That is, the cells are thought to be able to show two distinct phenotypes under identical culture conditions due to the diverse histories (induction) of gene expression.
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Here, two diverse biological functions leucine and histidine biosynthesis are designed, both of which result in a fitness recovery depending on the external conditions (Figure 5). For instance, it is the cells only showing an induced expression level of the “red” unit, which contacting the gene leuB essential for leucine biosynthesis that could survive under leucine-depleted conditions (i.e., Condition A). In general, Escherichia coli cells use the Leu operon and His operon to respond to starvation (Keller and Calvo, 1979; Searles et al, 1983; Wessler and Calvo, 1981). Depletion of leucine will lead to the induced expression of structural genes in the Leu operon; similarly, histidine depletion will cause an increase in expression of proteins encoded within the His operon (Henkin and Yanofsky, 2002; Keller et al, 1979). leuB and hisC, which are located within the Leu and His operons (Gama-Castro et al, 2008), are responsible for leucine and histidine biosynthesis, respectively. Rewiring these stringent starvation genes to the synthetic circuit not only introduces physiological activities to the synthetic design but also disturbs the original native regulation.
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3.2.2. Experimental investigation
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Firstly, whether the constructed synthetic circuit was functional was examined. The addition of IPTG induced the expression of the “red” unit that comprising leuB greatly improved the cell growth under the leucine-depleted conditions; similarly, the addition of doxycycline (Dox) induced the expression of hisC within the “green” unit, and allowed the cells to grow in histidine-depleted conditions (Table 2). Obviously, the selective full induction of the two expression units enabled the cells to grow under starved conditions. These results verified the following points: 1) the rewired genes, leuB and hisC, were biologically active, regardless of the chromosomal replacement; 2) the repressor-promoter interactions involved in the mutual inhibitory structure were strong; 3) the synthetic circuit was genetically stable and functional in the living cells.
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Table 2.
Cell growth and gene expression under varied conditions. +Leu, +His and Both AA indicate the addition of leucine, histidine and both amino acids, respectively. +IPTG, +Dox and No add. indicate the addition of IPTG, doxycycline, and in the absence of inducers, respectively. Cell growth is shown in the growth rate (h-1), and gene expression is marked as the induced gene name in red or green, indicating the fluorescence of the cell population.
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Figure 6.
Population shift in response to environmental transition. The distributions of newly formed populations (from 0 to 10 h or 33 h) are shown from light to dark grey, respectively. Green fluorescence intensity (GFP FI) and forward scattering (FSC) represent the abundances of GFP expressed in single cells and the relative cell size, respectively. GFP FI/FSC indicates the expression level of the “green” unit (i.e., hisC) in cells. The figures are partially modified from the original paper (Matsumoto et al, 2011).
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Subsequently, whether the synthetic circuit comprised of the rewired genes can be used by the Escherichia coli cells as a functional genetic switch in response to environmental transition was investigated. Under the hysteretic conditions, exponentially growing cells were transferred to the fresh media without the essential amino acid, leucine or histidine, for growth. Temporal changes in population dynamics were analyzed using flow cytometry, as described somewhere else (Matsumoto et al, in submission). When the “red” cell population of repressed expression of hisC was transferred to histidine-free conditions, the composition of the cell population changed gradually. More and more “green” cells of induced expression of hisC were born within the population, resulted in the population transition from “red” to “green” (Figure 6, left). Similarly, once the “green” cell population of repressed expression of leuB was transferred to leucine-free conditions, the “red” cells of induced expression of leuB were born and the fast growth allowed the “red” cells to take over the whole population, leading to the distribution shift from “green” to “red” (Figure 6, right). Note that in the rich medium, both “red” and “green” populations kept their distributions as initial expression level. Taken together, the Escherichia coli cells carrying this synthetic circuit formed the new population in accordance with the nutritional status.
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Furthermore, the relation between cell growth and expression of the synthetic circuit was evaluated. The temporal trajectory of the relation between cell growth and relative expression level in the cell population showed that the growth rate decreased significantly under conditions of leucine or histidine depletion but recovered gradually, accompanied by a gradual increase in leuB (“red” unit) or hisC (“green” unit) expression level. The growth recovery and population transition due to histidine depletion were faster than those due to leucine depletion. The “green” unit (hisC) was much easier to induce than the “red” unit (leuB), possibly due to slight leakage of gene expression from Ptet, diverse essentiality in amino acid requirement, or as yet unknown synchronised expression changes in other related genes, etc. Further applications using other genes and amino acids are required to determine the universality of the capacity of the synthetic circuit to respond to external perturbations and to function as genetic switch sensitive to surroundings.
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The experiments demonstrated that bacterial cells carrying this synthetic circuit formed diverse populations in response to the nutritional conditions and survived under conditions of nutrient depletion. A genome-integrated dual-function synthetic circuit sensitive to an environmental transition was successfully acquired. It strongly suggested that the synthetic design of proto-operons sensitive to external perturbations is practical for native cells. In summary, the method of genetic engineering and the application studies introduced here provides an efficient constructive approach for the studies or analysis in bacterial systems biology. Here, the genes involved in physiological functions responsive to external changes are introduced into the two expression units, the selective expression of the two units in cells can be considered a synthetic operon contributing to survival and/or adaptation.
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4. Conclusion
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A modified method for the integration of complicated genetic circuits into the Escherichia coli genome is introduced. The methodology provides an efficient synthetic approach for the dynamic and stochastic study of genetic networks. Linear artificial sequences as long as ~ 9 Kbps can be easily integrated into the bacterial genome at one time. The applications clearly showed accurate phenotypic behavior of the genome-integrated synthetic gene circuits corresponding to the design principle, which confirmed that the improved method allows the efficient construction of a single copy of a complicated genetic circuit in cells. As the genome recombination generally minimizes the copy number noise in the genetic circuit, it allows the precise design and interpretation of the cellular network. The availability of long-fragment insertions allows the easy reconstruction of complicated networks on the genome, and provides a powerful tool for synthetic and systems biology. Furthermore, the Escherichia coli cells carrying the synthetic circuit showed selective expression pattern in accordance with the environmental conditions, demonstrated the successful application of the genome-integrated synthetic circuit in cells. The applications owing to the simplified protocol demonstrated that the synthetic construct in the genome could be physiologically functional and sensitive to environmental transition. Synthetic approaches not only leads to the technical evolution for industrial use, but also can be employed to observe novel phenomena in living organisms. Further applications and improvement may bring us the completely synthetic genome functionally works in protocells.
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Acknowledgments
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We thank Natsuko Yamawaki, Junko Asada and Natsue Sakata for technical assistance, and the group members for fruitful discussion. This work was partially supported by Grants-in-Aid for Challenging Exploratory Research 22657059 (to BWY) and the “Global COE (Centers of Excellence) program” of the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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\n',keywords:null,chapterPDFUrl:"https://cdn.intechopen.com/pdfs/26402.pdf",chapterXML:"https://mts.intechopen.com/source/xml/26402.xml",downloadPdfUrl:"/chapter/pdf-download/26402",previewPdfUrl:"/chapter/pdf-preview/26402",totalDownloads:2060,totalViews:264,totalCrossrefCites:0,totalDimensionsCites:0,hasAltmetrics:0,dateSubmitted:"March 7th 2011",dateReviewed:"July 27th 2011",datePrePublished:null,datePublished:"January 20th 2012",readingETA:"0",abstract:null,reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/26402",risUrl:"/chapter/ris/26402",book:{slug:"advances-in-applied-biotechnology"},signatures:"Bei-Wen Ying and Tetsuya Yomo",authors:[{id:"77197",title:"Prof.",name:"Tetsuya",middleName:null,surname:"Yomo",fullName:"Tetsuya Yomo",slug:"tetsuya-yomo",email:"yomo@ist.osaka-u.ac.jp",position:null,institution:{name:"Osaka University",institutionURL:null,country:{name:"Japan"}}},{id:"77731",title:"Dr.",name:"Bei-Wen",middleName:null,surname:"Ying",fullName:"Bei-Wen Ying",slug:"bei-wen-ying",email:"ying@ist.osaka-u.ac.jp",position:null,institution:{name:"Osaka University",institutionURL:null,country:{name:"Japan"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Methodology: Genome-integration of foreign DNA sequences ",level:"1"},{id:"sec_2_2",title:"2.1. Refined method",level:"2"},{id:"sec_3_2",title:"2.2. High efficiency of recombination",level:"2"},{id:"sec_4_2",title:"2.3. Reduced cell-to-cell variation",level:"2"},{id:"sec_6",title:"3. Applications: Synthetic gene circuits work as design principles",level:"1"},{id:"sec_6_2",title:"3.1. Mutual inhibitory construct behaved as design principles",level:"2"},{id:"sec_7_2",title:"3.2. Synthetic switch sensitive to environmental transitions",level:"2"},{id:"sec_7_3",title:"3.2.1. Design principles",level:"3"},{id:"sec_8_3",title:"3.2.2. Experimental investigation",level:"3"},{id:"sec_11",title:"4. 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M.\n\t\t\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t2004\n\t\t\t\t\tLife history implications of rRNA gene copy number in Escherichia coli.\n\t\t\t\t\tAppl Environ Microbiol\n\t\t\t\t\t70\n\t\t\t\t\t6670\n\t\t\t\t\t6677 .\n\t\t\t'},{id:"B38",body:'\n\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tTsuru\n\t\t\t\t\t\t\tS.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tYasuda\n\t\t\t\t\t\t\tN.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tMurakami\n\t\t\t\t\t\t\tY.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tUshioda\n\t\t\t\t\t\t\tJ.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tKashiwagi\n\t\t\t\t\t\t\tA.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tSuzuki\n\t\t\t\t\t\t\tS.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tMori\n\t\t\t\t\t\t\tK.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tYing\n\t\t\t\t\t\t\tB. W.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tYomo\n\t\t\t\t\t\t\tT.\n\t\t\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t2011 Adaptation by stochastic switching of a monostable genetic circuit in Escherichia coli. Mol Syst Biol 7: 493.\n\t\t\t'},{id:"B39",body:'\n\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tWessler\n\t\t\t\t\t\t\tS. R.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tCalvo\n\t\t\t\t\t\t\tJ. M.\n\t\t\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t1981 Control of leu operon expression in Escherichia coli by a transcription attenuation mechanism. J Mol Biol\n\t\t\t\t\t149\n\t\t\t\t\t579\n\t\t\t\t\t597 .\n\t\t\t'},{id:"B40",body:'\n\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tYing\n\t\t\t\t\t\t\tB. W.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tIto\n\t\t\t\t\t\t\tY.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tShimizu\n\t\t\t\t\t\t\tY.\n\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\t\tYomo\n\t\t\t\t\t\t\t\n\t\t\t\t\t\t\n\t\t\t\t\t\n\t\t\t\t\t2010 (2010) Refined method for the genomic integration of complex synthetic circuits. J Biosci Bioeng\n\t\t\t\t\t110\n\t\t\t\t\t529\n\t\t\t\t\t536 .\n\t\t\t'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Bei-Wen Ying",address:null,affiliation:'
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Nikitina",authors:[{id:"159232",title:"Dr.",name:"Olga",middleName:null,surname:"Tsivileva",fullName:"Olga Tsivileva",slug:"olga-tsivileva"},{id:"166498",title:"Ms.",name:"Ekaterina",middleName:null,surname:"Loshchinina",fullName:"Ekaterina Loshchinina",slug:"ekaterina-loshchinina"},{id:"166499",title:"Dr.",name:"Valentina",middleName:null,surname:"Nikitina",fullName:"Valentina Nikitina",slug:"valentina-nikitina"}]},{id:"42585",title:"Role of Biotechnology for Protection of Endangered Medicinal Plants",slug:"role-of-biotechnology-for-protection-of-endangered-medicinal-plants",signatures:"Krasimira Tasheva and Georgina Kosturkova",authors:[{id:"161343",title:"Dr.",name:"Georgina",middleName:null,surname:"Kosturkova",fullName:"Georgina Kosturkova",slug:"georgina-kosturkova"},{id:"161344",title:"Dr.",name:"Krasimira",middleName:null,surname:"Tasheva",fullName:"Krasimira Tasheva",slug:"krasimira-tasheva"}]},{id:"40830",title:"The Use of Interactions in Dual Cultures in vitro to Evaluate the Pathogenicity of Fungi and Susceptibility of Host Plant Genotypes",slug:"the-use-of-interactions-in-dual-cultures-in-vitro-to-evaluate-the-pathogenicity-of-fungi-and-suscept",signatures:"Katarzyna Nawrot-Chorabik",authors:[{id:"107730",title:"Dr.",name:"Katarzyna",middleName:"Joanna",surname:"Nawrot-Chorabik",fullName:"Katarzyna Nawrot-Chorabik",slug:"katarzyna-nawrot-chorabik"}]}]}]},onlineFirst:{chapter:{type:"chapter",id:"64251",title:"Synthesis and Application of Porous Kaolin-Based ZSM-5 in the Petrochemical Industry",doi:"10.5772/intechopen.81375",slug:"synthesis-and-application-of-porous-kaolin-based-zsm-5-in-the-petrochemical-industry",body:'\n
\n
1. Introduction
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Access to a variety of energy sources has been fundamental in driving human development. Fossil fuels have been a major source of energy for mankind for more than 5000 years. Today, crude oil continues to be a significant contributor to the energy sector; it accounts for a large percentage of the world’s energy consumption. The production of chemicals has also continued to play a pivotal role in our daily activities. Interestingly, the amount of chemicals produced and used for both domestic and industrial purposes is very much related to the growth in global population. However, energy sources such as crude oil are non-renewable sources of fuel and current estimations show that world oil supplies will be depleted in the next century. Apart from the uncertainties in crude oil reserves, a major cause for concern is the impact crude oil extraction and its refining has on the environment; the combustion of fossil fuels leads to a net increase in greenhouse gases (GHG) leading to global warming. These concerns among others have led researchers in the recent past to explore alternative energy sources to the traditional crude oil for the production of fuels and petrochemicals. Various practises such as the use of efficient catalysts and augmented reactor technology are currently being employed towards ensuring that production technologies are eco-friendly and sustainable.
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Nanoporous materials are a large class of materials which consist of either an organic or inorganic framework structure containing ordered porous networks. They are generally classified by having pores sizes less than 100 nanometres and may be subdivided into three categories i.e. Microporous (<2 nm), Mesoporous (2–50 nm) and Macroporous (>50 nm). Their ability to interact or discriminate molecules depending on size has granted them scientific and technological importance. Research interest in nanoporous materials continues to grow as researches attempt to understand structure–property relations and design materials tailored for certain applications. Dependent on the properties of the nanoporous materials, applications range from purification and separation, sorption and drug delivery to energy storage, solar and fuel cells as well as electronic and magnetic devices. Typical examples include activated carbon, metal organic frameworks, ceramics, various polymers, aerogels, silicates and zeolites to name a few. Zeolites are microporous aluminosilicate materials that possess a 3-dimensional pore structure and play a prominent role in the petrochemical industry as ion exchangers, adsorbents, in separation and catalysis [1, 2, 3, 4]. Their shape selective properties permit control of product distribution in chemical reactions and as such have become indispensable catalysts in many petrochemical processes [5]. Of particular importance to the petrochemical industry is zeolite ZSM-5. ZSM-5 because of its unique channel structure, acidity, and hydrothermal stability has been used as a shape selective catalyst in isomerization, alkylation, oligomerisation and catalytic cracking reactions [6, 7, 8, 9]. It is conventionally synthesised using chemical sources such as sodium silicate solutions or silica gels and aluminium salts as the starting materials. Commercial synthetic zeolites are preferred over their naturally occurring analogues due to higher purity and uniform particle size which makes them more suitable for scientific and industrial applications [10]. However, zeolite synthesis using conventional methods leads to large amounts of waste being produced and chemical sources may be expensive, leading to high costs of zeolite production which limit commercialisation and use in many industrial applications [11]. Recently there have been increased efforts to explore the use of more affordable, natural raw materials possessing the necessary requirements for the synthesis of zeolites. ZSM-5 has been synthesised from natural silica and alumina sources such as rice husk ash [12, 13, 14], expanded perlite [15], palygorskite [16], fly ash [17], and kaolinite [18, 19, 20, 21]. The main drive to utilise these rich aluminosilicate minerals is their relative abundances, cost effectiveness and overall more environmentally friendly synthetic procedures. Although many natural minerals and manufacturing wastes have been utilised to synthesise a wide variety of zeolite structures, this chapter will focus on kaolin-based ZSM-5 synthesis and possible application in the petrochemical industry.
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Kaolin is a white clay composed mainly of kaolinite, a hydrous aluminosilicate mineral containing silica and alumina in a 1:1 ratio as well as impurities such as quartz and mica. Kaolin may require beneficiation to remove impurities depending on its application. Due to its low Si/Al ratio it has been extensively use in the synthesis of low silica zeolites [22, 23]. High silica zeolites such as ZSM-5 have also been synthesised with the addition of supplementary silica sources as well as through dealumination of kaolin via acid treatment [24, 25, 26]. Of the extensive range of aluminosilicate minerals used as zeolite precursors, kaolin has been favoured due to its ubiquitous nature. However, the studies of kaolin from different areas are significant since kaolin varies in composition depending on its geological occurrence. The chemical compositions of materials affect their properties and variations in the kaolin structure and composition can thus affect its subsequent chemical reactivity [27, 28].
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In this chapter the synthesis of kaolin-based ZSM-5 and the factors affecting synthesis are discussed. The work presented will focus on the synthesis of ZSM-5 using kaolin of South African origin. Most studies of kaolin derived ZSM-5 is performed on commercial kaolin. Only few have been done using raw kaolin. Chemical reactivities of kaolins obtained from different geological areas and the need to optimise synthesis conditions tailored to particular kaolin are highlighted. The effects of kaolinite content and synthesis parameters such as crystallisation time and temperature are discussed. The work is extended to include the effects of silica to alumina ratio on the physicochemical properties of ZSM-5 and is the main focus of this chapter. Furthermore, the application of kaolin-based ZSM-5 in important petrochemical processes such as the oligomerisation of olefins to fuel range hydrocarbons is evaluated.
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\n
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2. Kaolin in zeolite synthesis
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In the search for cheaper and more environmentally friendly alternatives to chemical sources, much research has been conducted on the feasibility of kaolin. By converting kaolin to the more reactive metakaolin via thermal activation and subjecting it to hydrothermal treatment in a NaOH medium, zeolite is produced. The use of kaolin as a source of silica and alumina was reported by Barrer [29] after it was calcined between 700 and 1000°C to form metakaolin. However due to the variations in kaolin composition and structure its subsequent chemical reactivity may be affected. Synthesis of zeolites from kaolin is affected by factors such as degree of crystallinity of the kaolin [23], kaolin composition [30], mineralogical impurities [23, 31], calcination temperature of kaolin [32], specific surface area of kaolin [33], synthesis parameters such as crystallisation temperature, time [30] and silica alumina ratio.
Many studies have been performed employing kaolin as the starting material for zeolite synthesis [34]. Investigations on the effects of kaolin crystallinity are contradictory as some researchers have shown that differences in the reaction kinetics of zeolite formation are observed for kaolin with different crystallinities or structural ordering [23] whereas others have reported that no significant differences were established when synthesising zeolites from kaolin of different crystallinity and reported that reactions of metakaolinites are independent of defects in the original crystal structure [22]. When two kaolins of South African origin from different geological areas i.e. Grahamstown (BK) and Fishoek (SK) were analysed it was shown that they differed in their structural order as well as composition and SK was more crystalline than BK [35]. The ZSM-5 synthesised using the two kaolin precursors resulted in differences in the crystallisation kinetics. The more disordered kaolin (BK) showed faster crystallisation kinetics than the more ordered SK. The physical and chemical properties of the reactive metakaolin of BK and SK were compared. The morphology obtained from SEM analysis shown that BK was composed of highly disordered loose kaolin plates compared to SK which possessed highly ordered stacked layers. The ordering remained even after calcination to form metakaolin. It was suggested that the highly disordered kaolin dissolved at a faster rate into the gel solution compared to SK in which the stacking layers were preserved. The chemical properties of the kaolin samples were analysed using 27Al and 29Si NMR MAS spectroscopy. The different coordination’s of aluminium and the relative amounts of each coordination type were correlated with the differences in reactivity of the two kaolins. These were determined for the kaolin before and after thermal activation, in the starting gels and products in order to observe the transformation of the Al environment as the zeolite was formed. After calcination of the precursors to form metakaolin, three distinct coordination’s of Al i.e. tetrahedral (AlO4), penta (AlO5) and octahedral (AlO6) species were observed however the relative amount of each differed between the two kaolins. The AlO6 in kaolin was converted to more reactive AlO5 and AlO4 sites via dehydroxylation of kaolinite [36]. Metakaolin SK contained a larger amount of AlO5 coordinated Al whereas metakaolin BK contained a larger amount of tetrahedrally coordinated Al at higher chemical shifts indicating the Aluminium existed in different environments of AlO4 coordination. The peak occurring at higher chemical shift in 27Al MAS NMR suggested the Al atoms are surrounded by different neighbouring atoms. Both q4 and q3 groups were identified. The q4 group has 4 neighbouring Si atoms and the q3 Al has 3 Si and 1 OH neighbouring group and is a highly reactive species [37]. It had been reported that metakaolins containing larger amounts of AlO4 species due to metakaolinisation at the optimal thermal activation temperature showed increased chemical reactivity. The optimum thermal activation temperature however differed for kaolins of different structural order [38]. In the case of BK and SK the thermal activation temperatures were identical. Therefore, the results suggested that the amount of reactive q3 AlO4 species in particular may influence kaolin chemical reactivity and that chemical reactivity of the kaolin is indeed related to its crystallinity when thermally activated at the same temperature. 29Si NMR also showed that Si atoms existed in different environments. Q2 and Q4 groups were identified. The Qn groups refer to the number of neighbouring Si atoms [39, 40]. The relative amount of Q2 and Q4 groups showed that metakaolin of SK possessed more Q4 (74%) species compared to metakaolin BK having more Q2 (79%) species. This indicated that the Al and Si environments of BK and SK were different after calcination at the same temperature. The higher amount of neighbouring Si atoms in SK confirmed a higher degree of networking between Si atoms. This may therefore be related to a higher degree in structural order which may affect dissolution of metakaolin into the gel and the hence the crystallisation kinetics. When the dried gels of the zeolite batch mixtures were analysed by 29Si NMR it was noticed that the gels were very similar and both exhibited a high degree of networking between Si atoms. However the 27Al NMR differed as shown inFigure 1.
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Figure 1.
27Al MAS NMR spectra of dried gels of BK and SK showing the presence of different tetrahedral species [35].
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The dried gel spectra of BK and SK both show broad peaks in the tetrahedral coordination range with peak maxima at ~55 ppm corresponding to q4 Al atoms. Dried gel BK however, possesses a shoulder peak at ~65 ppm indicating the presence of the reactive q3 Al species. Therefore the q3 Al from the calcined kaolin dissolves into the gel mixture and provides more reactive aluminium species to the starting gel of BK. The results also indicate that since the Si NMR spectra are similar the difference in chemical reactivity is mainly due to reactive alumina. The presence of the q3 Al species may quickly form aluminosilicate species and govern the incorporation of Al into the framework and is most likely responsible for the faster crystallisation kinetics observed in ZSM-5 derived from BK.
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The difference in crystallinity of kaolins and hence the difference in crystallisation kinetics leads to variations in the physicochemical properties of the zeolite. The synthesis parameters therefore have to be optimised to obtain a highly crystalline ZSM-5 zeolite. In this case for the more ordered SK a longer crystallisation time of 96 h compared to 48 h for BK was required to obtain a crystalline ZSM-5 with well-developed micropores. Furthermore, the acidic properties are also affected and ZSM-5 was noticed to increase in acidity with an increase in crystallisation time when the more ordered SK was used a precursor.
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2.1.2. Kaolinite content
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The composition of kaolin varies greatly depending on its formation process and also can affect the chemistry of the clay. The clay comprises mainly kaolinite as well as impurities such as quartz, muscovite and feldspars. Other contaminants such as iron oxide and titania may also be present. The purity of kaolin determines its use in a range of applications. Kaolin if properly processed could be utilised in the production of whiteware ceramics, paper and filler pigment [41, 42] as well as in catalysis and cement production [43]. Highly contaminated kaolins such as those containing high amounts of iron oxide are use in the manufacture of bricks. While most studies have focused on the use of pure commercial kaolin in the synthesis of zeolites, only a few studies on the use of raw or virgin kaolins have been reported. Synthesis of ZSM-5 and zeolite A from kaolin of Nigerian origin containing a high amount of quartz has been reported [20, 44]. The respective zeolites could be synthesised after using beneficiation techniques (i.e. extensive settling and flocculation) or a modified autoclave to separate impurities from the synthesis gel. However, the ZSM-5 final product still contained quartz and mordenite impurities attributed to unreacted metakaolin and similarities in the synthesis conditions for both ZSM-5 and mordenite. The zeolite A purity was affected by colloidal impurities in the dispersion and the ‘virgin’ kaolin still required some treatment before use.
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ZSM-5 was synthesised using highly impure kaolin with a high quartz content of large and finely grained quartz and muscovite originating from Grahamstown, South Africa [30]. Synthesis of ZSM-5 from raw (RK) and beneficiated kaolin (BK) was performed and the effect of the kaolinite content was investigated. Beneficiation was used to remove the majority of quartz and muscovite impurities and increase the kaolinite content. The composition of the kaolin was shown to play a significant effect on the formation of ZSM-5. From the beneficiated kaolin a highly pure crystalline ZSM-5 could be obtained. A higher kaolinite content results in more active silicate and aluminate species originating from the metakaolinite being present, which easily dissolve into the gel medium and form the primary building units necessary for nucleation and crystal growth in a shorter time period [21]. The results suggested higher kaolinite content shortens the induction period, increases the nucleation rate and hence the crystallisation of ZSM-5. However, from the raw kaolin ZSM-5 could only be synthesised under optimum synthesis conditions i.e. crystallisation temperature and time which was established for the particular kaolin during the study. Even under optimum conditions ZSM-5 synthesised from RK still contained small quartz impurities. The quartz impurities are difficult to dissolve and are therefore undesirable in kaolin [45]. Substantial amounts of alumina may also be required when kaolin is used in zeolite synthesis [46]. Thus, utilising kaolin with low kaolinite contents (i.e. alumina source) and high quartz content may result in the hindrance of ZSM-5 formation under certain crystallisation conditions. Furthermore, the results from this study suggested that beneficiation is a necessary step for the synthesis of pure ZSM-5.
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2.1.3. Crystallisation temperature and time
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The synthesis parameters of crystallisation temperature and time are critical in controlling the phase purity of ZSM-5. Temperature is a major factor in the formation of zeolites due to its strong effects on nucleation and crystal growth [47]. The effect of crystallisation temperature on ZSM-5 formation from BK was studied by holding the crystallisation time constant at shorter (24 h) medium (48 h) and longer times (96 h) and varying the temperature from 120 to 190°C [30]. The XRD diffractograms showed a large amorphous peak corresponding to amorphous aluminosilicate in the gel mixture is present at 120°C. As the synthesis temperature was increased to 150°C the amorphous gel is converted into pure, crystalline ZSM-5 phase as the nucleation rate increased. At 190°C the metastable ZSM-5 re-dissolves into the gel and the intensity of the ZSM-5 peaks decreases as a more thermodynamically stable quartz phase crystallises. At a high temperature of 190°C zeolite synthesis follows the Ostwald’s law of successive reactions. The initial metastable phase is replaced successively by more stable phases, in this case quartz. This trend is also observed at shorter and longer crystallisation times. Therefore at all crystallisation times studied, the optimum temperature for obtaining a highly crystalline pure ZSM-5 was determined to be 150°C. Similar results were reported by for the synthesis of ZSM-5 from Ahoko Nigerian Kaolin [48]. Although we might expect increased nucleation and crystal growth with an increase in temperature to 190°C, thermodynamic effects predominate over kinetic effects and favour the formation of the thermodynamically more stable quartz phase rather than growth of metastable ZSM-5 at 190°C.
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Crystallisation time is critical in controlling the crystallinity of the synthesised ZSM-5. It was also shown to have a major impact on the physicochemical properties such as porosity and morphology of ZSM-5. The relative crystallinity determined from XRD increased with time at optimum crystallisation temperature of 150°C. SEM results showed different morphologies and crystal sizes could be obtained by varying the crystallisation times between 24 and 96 h. ZSM-5 with high external surface area and both micro and mesopores were obtained at 150°C 24 h using BK and that micro-pore area increased with time as the relative crystallinity increased. The studies on the effects of crystallisation temperature and time clearly highlighted the need to determine optimum synthesis conditions to obtain pure well crystallised ZSM-5.
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2.1.4. SiO2/Al2O3 ratio
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In the synthesis of ZSM-5 the Si/Al ratio is known to affect physical properties such as crystal size and morphology as well as chemical properties such as acidity. The acid site density, type and strength are affected by the presence of aluminium and can be controlled by adjusting the Si/Al ratio. The acidity of the zeolite is important as ZSM-5 is used in many acid catalysed petrochemical reactions such as oligomerisation of olefins, cracking, isomerisation and alkylation. In this study the Si/Al ratios were varied and its effects on the physicochemical properties of the ZSM-5 are investigated. Raw kaolin (RK) and beneficiated kaolin (BK) are used as alumina source. The study shows the effects of the kaolin compositions when the Si/Al ratios are varied on the formation and properties of the zeolites. The ZSM-5 zeolites were synthesised using RK and BK with molar SiO2/Al2O3 ratios of 150, 70 and 42 in the batch mixture. The Si/Al ratios in the final product were determined using EDS atomic analysis. (It must be noted that the term Si/Al is used interchangeably but SiO2/Al2O3 is meant when referring to the batch ratio and Si/Al is meant when referring to the ratio in the product as determined by EDS analysis.) The atomic percentages of Si and Al are shown in Table 1.
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Zeolite
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Atomic % Si
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Atomic % Al
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Si/Al ratio
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\n\n\n
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Si-Al 150 RK
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11.67
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0.30
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39.0
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Si-Al 70 RK
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97.2
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2.77
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35.1
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Si-Al 42 RK
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91.51
\n
6.48
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14.1
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Si-Al 150 BK
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11.97
\n
0.30
\n
39.9
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Si-Al 70 BK
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42.18
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2.51
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16.8
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Si-Al 42 BK
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37.2
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5.04
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7.4
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Table 1.
EDS results showing atomic composition (%) of synthesised ZSM-5 with different Si/Al ratios using RK and BK.
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The Si/Al ratios of the final ZSM-5 product decrease as the initial Si/Al ratios in the batch mixtures decrease for both RK and BK. The Si/Al ratios of ZSM-5 synthesised from batch mixtures 70 and 42 BK are lower, approximately half that synthesised from RK of the same starting ratios respectively. This suggests that as the Si/Al ratio decreases less silica may be incorporated into ZSM-5 formed from BK compared to RK.
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The effect of Si/Al ratio on the structure of ZSM-5 was confirmed by x-ray diffraction. The diffractograms are shown in Figure 2(a) and (b). All patterns exhibited the characteristic ZSM-5 peaks at 7.94° (011), 8.90° (020), 23.10° (051), 24.0° (033) and 24.35° (313) 2θ. All samples synthesised from RK also showed a reflection at 26.67° 2θ corresponding to the presence of quartz. The intensity of this peak increased as the Si/Al ratio decreased. RK used as the aluminium source contains a significant amount of quartz. Samples synthesised with lower Si/Al ratios thus contained a larger amount of quartz which remained unreacted hence the increase in intensity of the quartz phase. As the Si/Al ratio in the RK samples decreased the intensity of the (011) and (020) reflections diminished and peaks shifted to slightly lower angles. This is due to a change in the crystal size and an increase in Al substitution in the framework structure. Peak intensity however increased from sample Si-Al 150 BK to Si-Al 70 BK as the material became more crystalline but then decreased in sample Si-Al 42 BK.
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Figure 2.
XRD powder patterns of ZSM-5 synthesised from (a) RK and (b) BK with different Si/Al ratios.
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The specific surface area and porosity characteristics of the samples are shown in Table 2 and N2 adsorption-desorption isotherms in Figure 3. From Table 2 it is noticed that the specific surface area increases as the Si/Al ratio decreases for the RK samples. The micropore, external surface area as well as the micropore volume increase as the Si/Al ratio decreases.
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\n\n
\n
Zeolite
\n
BET s/a m2/g
\n
Micropore area m2/g
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External area m2/g
\n
Avg. pore vol. m3/g
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Micropore vol. m3/g
\n
Avg. pore size nm
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\n\n\n
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Si-Al 150 RK
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214
\n
159
\n
55
\n
0.125
\n
0.073
\n
5.8
\n
\n
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Si-Al 70 RK
\n
253
\n
171
\n
82
\n
0.143
\n
0.079
\n
4.6
\n
\n
\n
Si-Al 42 RK
\n
274
\n
189
\n
85
\n
0.155
\n
0.087
\n
4.8
\n
\n
\n
Si-Al 150 BK
\n
223
\n
137
\n
86
\n
0.133
\n
0.063
\n
6.8
\n
\n
\n
Si-Al 70 BK
\n
340
\n
162
\n
178
\n
0.174
\n
0.072
\n
3.1
\n
\n
\n
Si-Al 42 BK
\n
265
\n
121
\n
144
\n
0.162
\n
0.055
\n
4.2
\n
\n\n
Table 2.
Specific surface area and porosity characteristics of RK and BK samples with different Si/Al ratios.
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Figure 3.
N2 adsorption-desorption isotherms of the representative samples.
\n
This indicates that the increase in aluminium content in the samples leads to a well-developed internal microporous structure. The increase in external surface area with lower Si/Al ratios is likely due to the decrease in crystal size and correlates with the decreased intensity of the XRD reflections. The presence of more quartz as the Si/Al ratio decrease may also result in a higher external surface area. All samples possess a type 1 adsorption isotherm indicative of the microporous nature of the synthesised ZSM-5. The samples also exhibit a H4 type hysteresis loop in the relative partial pressure range 0.4–1.0, indicating capillary condensation occurred. This has been attributed to the presence of a mesoporous phase associated with the slit shaped voids between packed crystals [49]. A large uptake in the P/Po range 0.9–1.0 suggests the presence of larger macropores. This however decreases with decrease in Si/Al ratio. The hysteresis loop becomes wider as the Si/Al ratio decreases. Similar results have been shown by Liu et al. [50] for ZSM-5 synthesised from gel mixtures of varying polymerisation degrees. The more depolymerised gel formed uniform aggregates of fine crystals. Therefore as the Si/Al ratio decreased the gel became more depolymerized forming aggregates of tiny crystals which when tightly packed form mesopores between them [51]. Si-Al 42 RK also showed a broad peak centred at approximately 12.4 nm in the pore size distribution curves shown in Figure 4(a) not seen in the other RK samples which is indicative of mesopores and hence the broader hysteresis loop. Samples from BK had a lower micropore area and volume and higher external surface area compared to those synthesised from RK. The crystallisation process is different when using RK and BK as the compositions differ. This may suggest a difference in the degree of polymerisation of silica and alumina in RK and BK. As suggested, the N2 isotherms and SEM images confirm the presence of aggregated nanocrystals in RK samples which form well depolymerized gel mixtures forming tetrahedral silica and alumina building blocks that form highly networked aluminosilicate species leading to a highly crystalline microporous structure. Zhang et al. [52] suggests a destruction of the microporous structure of ZSM-5 occurs with isomorphous substitution of Si with Al. This is possible as there is a higher content of aluminium in the BK samples compared to RK for each particular ratio. It is more likely however that extra-framework aluminium is present which blocks the micropores since Si-Al 70 BK has a higher micropore area and volume than Si-Al 150 BK but Si-Al 42 BK containing the highest aluminium content has the lowest microporosity. Therefore there is an optimum Si/Al ratio when synthesising ZSM-5 using BK. Interesting to note is the shape of the Si-Al 70 BK isotherms. It is a combination of a type I isotherm typical of ZSM-5 with a microporous framework and a type IV isotherm representative of mesoporous materials. The hysteresis loop in the 0.4–1.0 P/Po range is much smaller than other samples probably due to less space between packed crystals. However, the steep uptake at approximately 0.2 P/Po with a second small hysteresis loop may be due to the presence of uniform pores in the 2 nm range as shown in the pore size distribution graph for BK samples in Figure 4(b). Isotherms of this type have been reported in the literature for hierarchical zeolites [49, 53, 54, 55] and are ascribed to filling of mesopores with a narrow pore size distribution between 3 and 4 nm. The pore size in this sample is smaller and similar to the size of supermicropores described by Yang et al. [56]. From Figure 4(b) it is noticed that the presence of 2 nm sized pores increase with decreasing Si-Al ratio and exist even for Si-Al 42 BK which has the lowest Si-Al ratio although the amount decreased probably due to the blockage of pores by extra-framework aluminium. We consider that these are real pores and not physical phenomena observed in gas adsorption such as fluid to crystalline transitions observed in MFI zeolite structures on the basis that Si-Al 70 BK has a low Si/Al ratio. Fluid to crystalline effects are usually observed on high Si/Al ratio MFI and silicalite-1 materials due to the energetically homogenous surface created by the large amount of Si atoms which result in a well-pronounced sub-step in a narrow P/Po range and an increase in Al content induces energetic heterogeneity [57].
\n
Figure 4.
Pore size distribution calculated using the BJH method for the representative samples synthesised from (a) RK and (b) BK.
\n
HRSEM studies revealed that the morphology and particle size is affected by the change in Si/Al ratio. The images for the RK and BK samples are shown in Figure 5. Si-Al 150 RK has rounded boat shaped crystals that are highly inter-grown and agglomerated. There is also the presence of some amorphous material with no particular morphology.
\n
Figure 5.
High resolution SEM images showing morphology and crystal size of (a) Si-Al 150 RK, (b) Si-Al 70 RK, (c) Si-Al 42 RK, (d) Si-Al 150 BK, (e) Si-Al 70 BK and (f) Si-Al 42 BK.
\n
As the Si/Al ratio decreases the crystal size also decreases as shown in Figure 5(a–c). This agrees well with results reported in literature [24] and correlates with the increase in surface area and decrease of intensity of reflections as shown by BET and XRD results respectively. The material also becomes less inter-grown and aggregates with a uniform size distribution are noticed. Looking at the insets of Figure 5(b)and(c) it can be clearly seen that the aggregates are made up of nano-sized crystals that interlock and form an overall cross discus shape. ZSM-5 with similar morphology has been synthesised by Yue et al. [58] in the synthesis of hierarchical zeolites from kaolin and rectorite using a nanoscale depolymerisation-reorganisation approach and by Liu et al. [50] from highly depolymerised gel mixtures which also show similar trends in gas adsorption analysis to our samples. Thus an explanation of the change in morphology to aggregates of nano-crystals with a decrease in Si/Al ratio is that the gel mixture becomes more depolymerised with increased amount of the aluminium source. This trend is only observed in RK samples and not BK. The formation of the aggregates is due to the difference in composition of RK and BK in which the former contains a high quantity of quartz. As the aluminium amount is increased to obtain lower Si/Al ratios the quartz content in the gel mixture also increases and is present as shown in XRD. The quartz is difficult to dissolve which means that the alkalinity may be higher for the more soluble species i.e. that obtained from kaolinite and additional water-glass which is readily consumed. As crystallisation proceeds it becomes more depolymerized and leads to formation of fine crystals [50].
\n
Si-Al 150 BK had larger crystals than Si-Al 70 and 42 BK and followed the trend of decreasing crystal size with lower Si/Al ratios. As noticed in Figure 5(d) the morphology is hexagonally shaped crystals. Twinning is also apparent as the 100 face protrudes from the 010 face and is commonly seen in ZSM-5 crystal twinning. Si-Al 70 BK and Si-Al 42 BK both have wide crystal size distributions of both micrometre and sub-micrometre crystals that are highly intergrown with the former possessing smooth hexagonal crystals and the latter more rounded and rough edged crystals. Thus the Si/Al ratio also has an effect on morphology which agrees with work that has been reported in the literature [24].
\n
The amount and strength of acid sites were determined by the NH3 detected during desorption and the peak maximum in the desorption profile respectively. The results are summarised in Table 3.
\n
\n
\n
\n
\n
\n
\n
\n\n
\n
Samples
\n
Peak (°C)
\n
Acidity distribution (μmol NH3/g)
\n
\n
\n
Low temp
\n
High temp
\n
Weak
\n
Strong
\n
Total
\n
\n\n\n
\n
Si-Al 150 RK
\n
191
\n
395
\n
230
\n
225
\n
455
\n
\n
\n
Si-Al 70 RK
\n
192
\n
408
\n
269
\n
279
\n
548
\n
\n
\n
Si-Al 42 RK
\n
200
\n
421
\n
536
\n
500
\n
1036
\n
\n
\n
Si-Al 150 BK
\n
200
\n
413
\n
354
\n
279
\n
633
\n
\n
\n
Si-Al 70 BK
\n
210
\n
440
\n
689
\n
519
\n
1208
\n
\n
\n
Si-Al 42 BK
\n
203
\n
423
\n
584
\n
206
\n
790
\n
\n\n
Table 3.
Distribution of acidity on the ZSM-5 samples as determined by NH3-TPD.
\n
It is clearly seen from Table 3 that the total amount of acid sites increase with decreasing Si/Al ratio for RK samples as the higher the Si/Al ratio the lesser amount of total acid sites is [59]. This is true for the BK samples with the exception of Si/Al 42 BK which has the lowest Si/Al ratio but has lesser amount of acid sites than Si-Al 70 BK. This is most likely due to a larger amount of extra-framework aluminium and thus less Bronsted acid sites. This is supported by the fact that it has the least amount of strong acid sites (206 μmol/g), which are mainly due to NH3 desorption from Bronsted sites. The decrease in the amount and strength of acid sites when a batch ratio of 42 is used may be due to the aluminium atom being in close proximity to each other forming Al pairs which reduce the amount of acid sites. Both the amounts of weak and strong acid sites in general increase with decreasing Si/Al ratio. The RK samples have an almost 1:1 ratio of weak and strong acid sites. The BK samples however possess more weak acid sites than strong ones which may be due to NH3 desorption from the silanols caused by the defects in the crystalline structure. The strength of the acid sites varies with aluminium content and seems to increase with decreasing Si/Al ratio. The maxima of the peaks shift with decreasing Si/Al ratio to higher temperatures indicating that the NH3 molecule is more strongly bound to the acid sites. The maximum peak temperatures as shown in Table 3 indicate that ZSM-5 synthesised from BK have stronger acid sites when compared to those synthesised from RK with similar aluminium content. This may be due to the difference in aluminium environment as those synthesised from RK are more crystalline whereas those synthesised from BK contain more defects.
\n
It is well known that the aluminium content is directly related to the acidic properties of aluminosilicates. The presence of different Al species or coordination types leads to the formation of both Bronsted and Lewis acid sites. Therefore ZSM-5 samples with different Si/Al ratios synthesised from RK and BK were studied in order to determine the aluminium state, coordination, stability and degree of incorporation. The data obtained from 27Al MAS NMR investigations are presented in Table 4.
\n
\n
\n
\n
\n
\n\n
\n
Sample
\n
Al framework (% Alfr)
\n
Al extra-framework (% Alefr)
\n
PW at 1/2 max (~55 ppm Alfr)
\n
\n\n\n
\n
Si-Al 150 RK
\n
100
\n
n.d
\n
4.9
\n
\n
\n
Si-Al 70 RK
\n
92
\n
8
\n
5.5
\n
\n
\n
Si-Al 42 RK
\n
92
\n
8
\n
5.7
\n
\n
\n
Si-Al 150 BK
\n
93
\n
7
\n
5.3
\n
\n
\n
Si-Al 70 BK
\n
89
\n
11
\n
5.8
\n
\n
\n
Si-Al 42 BK
\n
83
\n
17
\n
5.6
\n
\n\n
Table 4.
Framework and extra-framework Al contents and line widths at half height for the representative ZSM-5 samples.
\n
Two peaks are commonly noticed in the 27Al NMR spectra of ZSM-5 zeolites. The major peak occurring at a chemical shift of approximately 55 ppm corresponding to the tetrahedrally coordinated Al in the zeolite framework and another smaller peak at approximately 0 ppm is related to the presence of octahedrally coordinated Al and is usually referred to as extra-framework aluminium [60]. This notion has been disputed, however, as evidence for octahedrally coordinated Lewis Al present as Al-(OSi)3(H2O)3 in the framework has been confirmed by Woolery et al. [61]. The relative amounts of the two species have been obtained by integration of the two peaks and are shown in Table 4. It is noticed that Si-Al 150 RK contains only tetrahedrally coordinated framework aluminium (Alfr) and any presence of extra-framework aluminium (Alefr) is negligible. It is common for highly siliceous ZSM-5 to possess only Alfr. Si-Al 150 BK which has a similar Si-Al ratio as Si-Al 150 RK as shown in Table 1 does have a small amount of Alefr. This may be due to the difference in the composition and hence solubility of RK and BK. A slight increase in Alefr is seen as the aluminium content increases for RK samples although there is not much difference between Si-Al 70 and Si-Al 42 RK. The increase in Alefr with increase in aluminium content however, is drastically enhanced for the BK samples. EDS analysis shows that Al content is almost double in Si-Al 70 and Si-Al 42 BK compared to that synthesised from RK with the same staring gel ratios confirming that more Al is incorporated into ZSM-5 from BK due to a more abundant supply of reactive aluminium. However, NMR suggests that the Al is not necessarily all in framework positions and Al available from RK is better incorporated into the framework structure as it has a greater amount (92%) compared to Si-Al 70 BK and Si-Al 42 BK only having 89 and 83% Alfr respectively. This result correlates with that obtained from N2 physisorption and SEM analysis which showed a larger micropore area and nano crystal aggregates respectively for the RK samples compared to BK. This suggests a well-structured crystalline material made up of tetrahedrally coordinated Si and Al. It must be noted that quantitatively the BK samples form more Al-rich ZSM-5 even though the Alefr amounts are greater. BK samples had a large external surface area greater than its micropore area due to mesoporosity. Larger amounts of aluminium may therefore be present at the external surface of the zeolite in a less stable octahedral form as suggested by Serrano et al. [53] for materials with more than 1 type of porosity such as hierarchical zeolites. They may also be present in different environments with more than 1 type of neighbouring atom as the resonances observed are broad and Si-Al 42 BK also shows a shoulder peak at ~6 ppm as shown in Figure 6.
\n
Figure 6.
27Al MAS NMR spectra of the RK and BK samples with different Si/Al ratios.
\n
From the 27Al NMR spectra of the RK and BK samples, The RK samples and Si-Al 150 BK all have a peak max at ~55.7 ppm. Si-Al 70 BK shows a slight shift to a lower value ~54 ppm and Si-Al 42 BK a larger shift to ~58.3 ppm. The peak width values shown in Table 4 are an indication of the uniformity of the Al environments in zeolite materials [53, 62] and crystallinity [63]. Si-Al 150 RK has the smallest peak width indicating high crystallinity and correlates with the XRD results and a more uniform Al environment. The peak width increases for all other samples indicating different environments of Al. The base of the peak begins at ~50 ppm and ends at ~62 ppm for all samples except Si-Al 42 BK which shows a shift of approximately ~4 ppm towards higher ppm values. Previous studies by Dedecek et al. [37] have shown that different environments for AlO4 tetrahedra exist after deconvolution and simulation of the tetrahedral peak. Peaks observed at 50, 53, and 58 ppm correspond to Al atoms with only Si neighbours, while resonances at 62 ppm corresponds to Al atoms with 3 Si and 1 neighbouring OH group which are highly reactive. Thus the resonances fall within the range of the peak observed for the ZSM-5 samples. The resonances at 50, 53 and 58 ppm reflect the difference in environment of the AlO4 tetrahedra in the sample which is due to the effect of the vicinity of second Al atom in the sample [37]. The closeness of the next Al atom in sequences such as Al-O-(Si-O)2-Al can change the observed shift of the Al atom by ~4 ppm. Thus the peak observed for Si-Al 42 BK at 58.3 ppm suggests that the Al atoms are in close proximity to each other and has been reported for Al rich zeolites [37, 64]. Si-Al 42 BK also more likely has more Al atoms with a neighbouring OH which are highly reactive as the resonance shifts towards 62 ppm. Therefore it is possible that BK forms more Al rich ZSM-5 than RK for two reasons. The first being that RK causes a higher degree of depolymerisation due to the presence of quartz (as discussed above) of the remaining Na-silicate which is broken down to silicate species. This then forms more networked silicate and aluminosilicate and has less formation of close Al atoms resulting in higher ratios. Second for the BK samples, which were prepared with a smaller amount of additional Na-silicate, was not well depolymerised due to a lower alkalinity i.e. the silicate remained non-transformed and balanced by the Na+ and only a much lower concentration of Si was available for the highly reactive Al species resulting in aluminosilicate with a much lower Si/Al ratio having also more Alefr and in the case of Si-Al 42 BK, in a high probability of aluminosilicate species with close Al atoms [37].
\n
\n
\n
\n
\n
3. Potential application of kaolin-based ZSM-5
\n
\n
3.1. Catalytic oligomerisation
\n
The performance of the zeolites as catalysts was tested in the transformation of 1-hexene, used as a model compound for the oligomerisation of alkenes; reaction was performed at the following conditions: T = 350°C, pressure = 1 atm, WHSV = 8 h−1. The activities of the catalysts were determined by assuming all 1-hexene present in the product was due to unconverted feed. The graph showing activity as a function of time on stream is shown in Figure 7.
\n
Figure 7.
Graph showing hexane conversion over different catalysts (P = 1 atm, T = 350°C, WHSV = 8 h−1).
\n
The catalytic tests were conducted over a time period of 420 min. All catalysts showed some deactivation over the total time on stream although some were more rapidly deactivated than others. All catalysts had a conversion not less than 90% in the first hour on stream. The highest conversion achieved was approximately 98%. It is clearly noticed that the conversion of the catalysts is highly dependent on the acidity, in particular the Bronsted acidity. Si-Al 42 RK and Si-Al 70 BK with similar acidities and possessing the highest acidities of the catalysts had the highest conversions and both showed only slight deactivation as the conversion remained above 90% for the duration of the reaction. Si-Al 70 RK and Si-Al 150 BK having identical amounts of strong acid sites and tetrahedral alumina (Tables 3 and 4 respectively) have the same conversion and deactivation for the time on stream. Si-Al 42 BK which had the least amount of Bronsted sites showed the lowest conversion and the most rapid deactivation. Interestingly, Si-Al 150 RK having a much lower acidity compared to Si-Al 42 RK and Si-Al 70 BK has a very similar activity and shows even better stability (Figure 7). This was attributed to the effect of quartz coating the surface of the ZSM-5 which prevents deactivation. Therefore it is seen that this catalyst functions better than catalysts with more than double its acidity and further validates the influence of quartz on the performance of the catalyst.
\n
The catalysts tested in the transformation of 1-hexene all possess a wide product distribution. The products are grouped accordingly: (C2–C5) range, (C6–C9) gasoline range and (C10+) diesel range. This indicates multiple types of reactions may occur at the set reaction conditions such as oligomerisation, cracking, isomerisation and alkylation to name a few. The selectivities to these ranges however change with time on stream and it is noticed that small changes in conversion can have large changes in selectivity to products. The selectivities of the catalysts are discussed further and correlated with their physicochemical properties.
\n
Si-Al 150 BK and Si-Al 70 RK are compared to each other since they have similar acidities as well as conversions over time on stream as shown in Figure 7. The selectivities however differ from each other. The selectivities to gasoline (C6–C9) and diesel (C10+) ranges are shown in Figure 8(a) and (b) respectively.
\n
Figure 8.
Selectivity of Si-Al 150 BK and Si-Al 70 RK to gasoline (C6–C9) (a) and diesel (C10+) (b) range products over time on stream.
\n
Both catalysts have a similar trend to selectivity of diesel range products as noticed in the graph and decrease with time as the conversion decreased. However Si-Al 150 BK has a slightly higher selectivity throughout the reaction. Both show good selectivity (40–55%) to gasoline products with the selectivity of Si-Al 70 RK possessing a steady increase over the reaction time. Si-Al 150 BK was more unstable and the selectivity varied over time and was slightly less than Si-Al 70 RK. It was thought Si-Al 70 RK was more selective to reactions pertaining to chain growth such as oligomerisation and alkylation but a closer look at the product selectivity of the gasoline range revealed that Si-Al 70 RK was highly selective to C6 hydrocarbons as compared to Si-Al 150 BK as shown in Figure 9. An increase from ~12% to above 30% was observed. The C2–C5 selectivity trend is also shown and decreases over time for the Si-Al 70 RK catalyst but shows an overall increase in the C2–C5 products for Si-Al 150 BK. Thus there may be a greater selectivity to isomerisation reactions over cracking reactions for the Si-Al 70 RK catalyst whereas Si-Al 150 BK has a better selectivity to chain growth reactions as its total selectivity to C7+ hydrocarbons is higher. This may suggest a slight difference in strength of acid sites available as oligomerisation and cracking occur on stronger acid sites than isomerisation reactions which occur on sites of intermediate acidity [65]. The main difference between the two catalysts however is the crystal morphology and size. The Si-Al 70 RK possessing nanocrystals as shown in HRSEM, this may reduce the effects of diffusion limitation. As shown by Buchanan et al. [66] larger crystals showed a higher selectivity to C3/C4 products and a lower isomerization/cracking of olefin ratio due to diffusion limitation. Therefore it is possible that as the strength of the acidic sites are reduced over time due to deactivation which happens more in Si-Al 70 RK, isomerization reactions which occur faster than cracking reactions [66], increase and due to the nanocrystals, the isomers which form are able to diffuse out before any secondary cracking leading to more C6 isomers, less cracked C2–C5 products and a better gasoline selectivity. Therefore the physical properties of the catalyst synthesised from RK are shown to affect its catalytic performance.
\n
Figure 9.
Selectivity of Si-Al 150 BK and Si-Al 70 RK to C6 (black curves) and C2–C5 (blue curves) range products over time on stream.
\n
Figure 10 shows the selectivity of Si-Al 150 RK, Si-Al 42 RK, Si-Al 70 BK and Si-Al 42 BK to diesel and gasoline range hydrocarbons. The three catalysts with similar conversions i.e. Si-Al 150 RK, Si-Al 42 RK and Si-Al 70 BK can be compared in terms of selectivity.
\n
Figure 10.
Selectivities to diesel range (C10+) and gasoline range (C6–C9) hydrocarbons of catalysts Si-Al 150 RK, Si-Al 42 RK, Si-Al 70 BK and Si-Al 42 BK as a function of time.
\n
From Figure 10 it is noticed that after the first hour on stream the selectivity to C10+ is the highest for the catalyst Si-Al 42 RK showing ~16% selectivity. This may indicate that oligomerisation to long chain hydrocarbons largely depends on the acidity since Si-Al 42 RK which has a high Bronsted acidity had the highest selectivity. The more acidic catalysts have better selectivity over the first 3 h on stream. As the reaction proceeds however, all catalysts show a large decrease in selectivity to C10+ hydrocarbons except Si-Al 150 RK which shows a greater stability. The decrease for Si-Al 42 RK is almost linear over the reaction time and drops to ~3%. The decrease in selectivity to C10+ suggests the deactivation of certain acid sites which are most likely strong Bronsted acid sites as oligomerisation reactions are favoured at these catalytic centres [65]. Si-Al 70 BK which has similar acidity to Si-Al 42 RK shows a slightly better selectivity to C10+ hence less deactivation of Bronsted sites. This is most likely due to the higher surface area and increased mesoporosity which may inhibit the build-up of carbonaceous deposits usually responsible for deactivation and hence further highlights the effects of the physicochemical properties of the zeolites on its catalytic behaviour. As mentioned in our previous work [30], the effect of quartz deposits on the external surface of the crystals in Si-Al 150 RK plays a major role in inhibiting the formation of carbonaceous material causing deactivation. Thus even when compared to catalysts with greater acid site density and strength (almost double its acidity), Si-Al 150 RK possesses greater stability and selectivity is almost double that of the more acidic catalysts. This clearly suggests that the acid sites are prevented from being deactivated by the deposition of quartz and continue to catalyse oligomerisation reactions for the duration of the reaction. Si-Al 42 BK had the lowest selectivity to C10+. This is most likely due to its low Bronsted acidity but also the high content of extra-framework aluminium may cause pore blockage and prevent access to acid sites for oligomerisation. The closeness of Al atoms in the structure may also have an effect on the acidity and types of catalytic reactions that are favoured.
\n
All catalysts showed good selectivity to the gasoline range. Si-Al 42 RK shows the opposite trend for selectivity to gasoline as this increase over the reaction time. The selectivity spikes in the last 2 h from 45 to 74%. Figure 11 shows the selectivity to C6 and C2–C5 products.
\n
Figure 11.
Selectivity to C6 hydrocarbons and C2–C5 selectivity of Si-Al 42 RK (blue curve) as a function of time.
\n
The spike in gasoline activity is again due to an increase in C6 selectivity. This further confirms our observations with the Si-Al 70 RK catalyst. The decrease in C10+ selectivity indicates deactivation of strong acid sites. Thus isomerisation reactions are then favoured when only less acidic site are available and due to Si-Al 70 RK and Si-Al 42 RK possessing the same morphology the isomerisation products diffuse out before cracking. Figure 11 clearly shows the contrasting selectivities between isomerisation (C6) and cracking reactions (C2–C5) for the Si-Al 42 RK catalyst. Si-Al 42 BK also shows good isomerisation activity which further indicates the reaction occurring on weaker acid sites.
\n
\n
\n
\n
4. Conclusion
\n
In conclusion, this chapter discusses some of the key factors affecting the synthesis of kaolin-based ZSM-5 such as kaolin crystallinity, kaolinite content, crystallisation parameters and Si/Al ratio. Additionally, the catalytic performance of the ZSM-5 derived from kaolin was evaluated. These factors are important considerations when attempting to synthesise ZSM-5 with high purity and crystallinity. However, ZSM-5 can also be synthesised from impure kaolin sources and these impurities may act as poisons or promoters during synthesis and catalytic application. The requirements to develop synthesis conditions that are optimised for specific sources of kaolin are established. The physicochemical properties such as porosity, morphology and acidity of ZSM-5 can be controlled by choosing the right synthesis procedures. Kaolin-based ZSM-5 zeolites are promising as catalysts for petrochemical reactions such as oligomerisation. High activity and selectivity to gasoline and diesel range hydrocarbons was attainable. The activity of the catalysts correlated well with the acidity of ZSM-5 samples. The catalytic performance of the zeolites also correlated well with the physical properties such as morphology and surface area which were shown to influence selectivity to certain products by favouring isomerisation and oligomerisation reactions respectively. Impurities in the kaolin precursor may also have positive effects on catalytic performance, in this case quartz deposition on ZSM-5 inhibiting deactivation and increasing catalyst stability. Therefore, ZSM-5 zeolites can be successfully synthesised from cheaper, more environmentally friendly alternative starting materials that have satisfactory performances in catalytic application.
\n
\n
Acknowledgments
\n
The authors would like to thank the Petroleum, Oil and Gas Corporation of South Africa (PetroSA) for their financial support and technical discussions, the National Research Foundation (NRF) for granting Ebrahim Mohiuddin a scholarship, Makana Brick for supplying the raw kaolin, the electron microscope unit, Physics department, University of the Western Cape for the SEM images, Mrs. E. Antunes, Chemistry department, University of the Western Cape and Dr. D.J. Brand, University of Stellenbosch for the solid state NMR work and Ithemba labs for the XRD work.
\n
\n',keywords:"kaolin, ZSM-5, synthesis, oligomerisation, cracking",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/64251.pdf",chapterXML:"https://mts.intechopen.com/source/xml/64251.xml",downloadPdfUrl:"/chapter/pdf-download/64251",previewPdfUrl:"/chapter/pdf-preview/64251",totalDownloads:259,totalViews:0,totalCrossrefCites:0,dateSubmitted:"June 14th 2018",dateReviewed:"September 7th 2018",datePrePublished:"December 5th 2018",datePublished:null,readingETA:"0",abstract:"Zeolites are advanced chemical materials that play a significant role in many petrochemical applications. In recent years, research interest in improving and enhancing the effectiveness of ZSM-5 as a catalyst has grown immensely. In particular, finding cheaper, environmentally friendly alternative starting materials for the synthesis of ZSM-5 has gained much attention. Kaolin has been widely investigated as a zeolite precursor as it comprises the required constituents for an aluminosilicate zeolite material; ubiquitous nature and its benefit in synthesising zeolites are well known as an inexpensive way of obtaining catalysts. This chapter deals with the factors affecting ZSM-5 synthesis when utilising a kaolin precursor. The effects of kaolin crystallinity, kaolinite content and synthesis parameters on ZSM-5 formation and its physicochemical properties are discussed. The potential of kaolin-based ZSM-5 as an oligomerisation catalyst is investigated. Pure, crystalline ZSM-5 could be successfully synthesised from a kaolin precursor. Physicochemical properties such as morphology, porosity and acidity are affected by the kaolin precursor and optimum synthesis conditions are required for synthesis of ZSM-5 from particular kaolin. Kaolin-based ZSM-5 catalyst showed good activity and selectivity to valuable fuel range hydrocarbons.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/64251",risUrl:"/chapter/ris/64251",signatures:"Ebrahim Mohiuddin, Yusuf Makarfi Isa, Masikana M. Mdleleni and David Key",book:{id:"7774",title:"Nanofluid Flow in Porous Media",subtitle:null,fullTitle:"Nanofluid Flow in Porous Media",slug:null,publishedDate:null,bookSignature:"Dr. Mohsen Sheikholeslami Kandelousi, Dr. Sadia Ameen, Dr. M. Shaheer Akhtar and Prof. Hyung-Shik Shin",coverURL:"https://cdn.intechopen.com/books/images_new/7774.jpg",licenceType:"CC BY 3.0",editedByType:null,editors:[{id:"185811",title:"Dr.",name:"Mohsen",middleName:null,surname:"Sheikholeslami Kandelousi",slug:"mohsen-sheikholeslami-kandelousi",fullName:"Mohsen Sheikholeslami Kandelousi"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. 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Journal of Catalysis. 2014;319:200-210\n'},{id:"B59",body:'Rodríguez-González L, Hermes F, Bertmer M, Rodríguez-Castellón E, Jiménez-López A, Simon U. The acid properties of H-ZSM-5 as studied by NH3-TPD and 27Al-MAS-NMR spectroscopy. Applied Catalysis A: General. 2007;328(2):174-182\n'},{id:"B60",body:'Cabral de Menezes SM, Lam YL, Damodaran K, Pruski M. Modification of H-ZSM-5 zeolites with phosphorus. 1. Identification of aluminum species by 27Al solid-state NMR and characterization of their catalytic properties. Microporous and Mesoporous Materials. 2006;95(1-3):286-295\n'},{id:"B61",body:'Woolery GL, Kuehl GH, Timken HC, Chester a W, Vartuli JC. On the nature of framework Brønsted and Lewis acid sites in ZSM-5. Zeolites. 1997;19(4):288-296\n'},{id:"B62",body:'Oldfield E, Haase J, Schmitt KD, Schramm SE. Characterization of zeolites and amorphous silica—Aluminas by means of aluminum-27 nuclear magnetic resonance spectroscopy: A multifield, multiparameter investigation. Zeolites. 1994;14(2):101-109\n'},{id:"B63",body:'Prasad JV, Rao KV, Bhat YS, Halgeri AB. MAS NMR studies of ZSM-5 zeolites: Correlation to para selectivity and SEM observations. Catalysis Letters. 1992;14(3-4):349-357\n'},{id:"B64",body:'Smaihi M, Barida O, Valtchev V. Investigation of the crystallization stages of LTA-type zeolite by complementary characterization techniqnes. European Journal of Inorganic Chemistry. 2003;24:4370-4377\n'},{id:"B65",body:'Van Donk S, Bitter JH, De Jong KP. Deactivation of solid acid catalysts for butene skeletal isomerisation: On the beneficial and harmful effects of carbonaceous deposits. Applied Catalysis A: General. 2001;212(1-2):97-116\n'},{id:"B66",body:'Buchanan JS, Olson DH, Schramm SE. Gasoline selective ZSM-5 FCC additives: Effects of crystal size, SiO2/Al2O3, steaming, and other treatments on ZSM-5 diffusivity and selectivity in cracking of hexene/octene feed. Applied Catalysis A: General. 2001;220(1-2):223-234\n'}],footnotes:[],contributors:[{corresp:null,contributorFullName:"Ebrahim Mohiuddin",address:null,affiliation:'
PetroSA Synthetic Fuels Innovation Centre, South African Institute for Advanced Materials Chemistry, University of the Western Cape, South Africa
PetroSA Synthetic Fuels Innovation Centre, South African Institute for Advanced Materials Chemistry, University of the Western Cape, South Africa
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