Science Journal Ubon Ratchathani University · Science Journal Ubon Ratchathani University Faculty of Science Ubon Ratchathani University Ubon Ratchathani 34190 THAILAND ISSN : …

Science Journal Ubon Ratchathani University

http://scjubu.sci.ubu.ac.th Faculty of Science Ubon Ratchathani University Ubon Ratchathani 34190 THAILAND ISSN : 1906-9294

Volume 1 Number 2 July-December, 2010

Copyright 2010 Faculty of Science, Ubon Ratchathani University. All Rights Reserved.

Advisory Editorial Board

David J. Lurie University of Aberdeen, UK

Hyoung Tae Choi Kangwon National University, Korea

David Ruffolo Mahidol University, Thailand

Chidchanok Lursinsap Chulalongkorn University, Thailand

Pranom Chantaranothai Khon Kaen University, Thailand

Sompong Dhampongsa Chiang Mai University, Thailand

Somsak Pantuwatana Burapha University, Thailand

Vichai Reutrakul Mahidol University, Thailand

Supot Hannongbua Chulalongkorn University, Thailand

Suthat Yoksan Srinakharinwirot University, Thailand

Sukit Limpijumnong Suranaree University of Technology, Thailand

Supa Hannongbua Kasetsart University, Thailand

Bhiyayo Panyarjun Chulalongkorn University, Thailand

Kosin Chamnongthai King Mongkut's University of Technology, Thailand

Naiyatat Poosaran ChiangMai University, Thailand

Somchit Chotchaisathit Khon Kaen University, Thailand

Santi Maensiri Suranaree University of Technology, Thailand

Vinich Promarak Ubon Ratchathani University, Thailand

Janpen Intaraprasert Ubon Ratchathani University, Thailand

Sonthi Kochawat

Ministry of Natural Resources and Environment, Thailand


Working Group

Consultant Dean, Faculty of Science, Ubon Ratchathani University Editor in Chief Chittakorn Polyon

Associate Editors in Chief Pongsak Rattanachaikunsopon Udom Tipparach

Pornpan Pongpo

Editors Anucha Yangthaisong Chan Inntam

Nongkhran Sasom

Parichat Phumkhachorn

Saisamorn Lumlong

Sungwan Kanso

Tinnagon Kaewin

Language Editors Bob Tremayne Ian Thomas

Managing Staff Amornrat Wasuree

Apinya Pitaksa

Dutruthai Sahapong Jiraporn Thongsud

Muntana Tonpun Nanthana Pimpan

Tutiyaporn Weerakul Wanwisa Songserm

Mewadee Noidee Art Work Surasit Sutthikhampa


I

Editorial

This is the 1st volume (Number 2, July-December, 2010) of Science Journal Ubon Ratchathani University (SCJ) published by Faculty of Science, Ubon Ratchathani University. In this volume, there are 11 research articles and a review article with totally 83 pages. The most content of the volume consists of research articles and the review article, involving with researches in the fields of chemistry, materials science, physics, statistics, biochemistry and biotechnology. We would like to take this opportunity to express our deep appreciation to all authors and reviewers who for their significant contributions to make SCJ happen. We also hope that authors who submitted their manuscripts to SCJ will get a taste of achievement in publishing in an international journal. In the future, we aim to strive for the best quality in all published articles.

Editorial Team

Science Journal Ubon Ratchathani University


II

Guide for Authors

The Science Journal Ubon Ratchathani University (SCJ) is a peer reviewed journal publishing high quality articles. The SCJ accepts all articles dedicated to all aspects of sciences such as physics, chemistry, materials science, biology, biochemistry, biotechnology, microbiology, environmental science, and other basic sciences such as education science, mathematics, statistics and computer science, including information technology. The articles required are either research articles or review articles, is defined as follows:

1. Review articles: an article which aims to present comprehensively already existing finding.

2. Research articles: a regular article which aims to present new findings.

The SCJ considers only manuscripts that have not been published (or submitted simultaneously) at any language, elsewhere. The SCJ is issued both in electronic form (for free) and printed form as two volumes per year (free for the authors).

The manuscript templates for both two article types should be prepared in english form, which can be downloaded from the SCJ homepage (http://scjubu.sci.ubu.ac.th). In the manuscript preparation, the article pages, including tables, figures and references, should be not over 12 pages. The sizes of figures in each article should be surely given high resolution enough to be clearly reviewed. The figures should be also used only in black-and-white. However, these figures could be prepared to be shown in colors in the online edition.

Each article in the SCJ is reviewed by two or three reviewers. The review process is about 2-3 weeks. The revision article should be revised in 1-3 weeks, depending on each recommendation from a reviewer. The average reviewed, revised and managed time for each article published in the SCJ is about 2-3 months.

In order to submit articles, authors should firstly register to obtain a username, and then login at the SCJ homepage (http://scjubu.sci.ubu.ac.th) to access the submission process. All information about the submitted articles would be informed by the SCJ editors ([email protected]) via e-mail of corresponding authors.


Contents Editorial I Guide for Authors II Emerging Trends of Natural-Based Polymeric Systems for Drug Delivery in Tissue Engineering Applications

1-13

M.S. Hasnain et al. Nano-Sized Titanium Dioxides as Photo-Catalysts in Degradation of Polyethylene and Polypropylene Packagings

14-20

T. Manangan et al. Effect of Zinc Oxide on the Morphology and Mechanical Properties of Poly(Styrene-co-Acrylonitrile)/Poly(Methyl Methacrylate)/Zinc Oxide Composites

21-26

S. Wacharawichanant et al. Study of Carbonyls-TiO2 as Co-Catalysts in Photo-Oxidative Degradation of Hydrocarbon Backbone

27-34

T. Manangan et al. Characteristics of TiO2-SiO2 Microparticle Composites Using Different Types of SiO2 Particle

35-39

J. Janlamool et al. Synthesis of Carbon Microspheres from Starch by Hydrothermal Process 40-45 S. Ratchahat et al. Immobilization of Lipase on CaCO3 and Entrapment in Calcium Alginate Bead for Biodiesel Production

46-51

N. Sawangpanya et al. Phytoremediation : Vetiver Grass in Remediation of Soil Contaminated with Trichloroethylene

52-57

J. Janngam et al. Risk Estimation of Campylobacter jejuni Caused by Chicken Meat Consumption for High Risk Group in Thailand

58-64

S. Osiriphun et al. Preparation of Purified Dye Powder from the Bark of Livistona speciosa 65-70 P. Muangthai et al. Continuous Cheese Whey Fermentation in a Series of Two Reactors 71-77 R. Agustriyanto et al. Image Structure of Large Dams in Kanchanaburi, Thailand, Using Surface GPR Reflection Techniques

78-83

S. Yooyuanyong et al.


Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 1-13 SCIENCE JOURNAL Ubon Ratchathani University http://scjubu.sci.ubu.ac.th

*Corresponding author. E-mail address: [email protected]

Review Article

Emerging Trends of Natural-Based Polymeric Systems for Drug Delivery in Tissue Engineering Applications

M.S. Hasnain1, A.K. Nayak2*, F. Ahmad3, R.K. Singh1 1Department of Pharmaceutical Chemistry, Seemanta Institute of Pharmaceutical Sciences,

Mayurbhanj-757086 Orissa, India. 2Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences,

Mayurbhanj-757086. Orissa, India. 3Department of Clinical Research, Jamia Hamdard, New Delhi-110062, India.

Received 7/09/10; Accepted 13/12/10

1. Introduction

From last few decades, an impressive progress has been recorded in terms of developing new materials or refining existing material composition or microstructure in order to obtain better performance for drug delivery in various tissue engineering appli-cations. The growing demand of tissues and organs for transplantation and inability to fulfill this need using both autogenic (from

the host) and allogeneic (from the same species) sources has led to rapid advancement of tissue engineering as an option. The term ‘tissue engineering’ was officially defined in a National Science Foundation (NSF) workshop (USA) in 1988 as “the application of principles and methods of engineering and life sciences toward fundamental under-standing of structure-function relationships in normal and mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function” [1]. Tissue engineering can help to improve the quality of healthcare in a wide range of

Abstract

Tissue engineering is the most promising therapeutic approach, in which damaged or diseased tissues are regenerated by using different biomaterials with or without various drug molecules like antibiotics, proteins, growth factors etc. Different biomaterials have been proposed for drug delivery in tissue engineering applications. Among them, natural occurring polymers are the most attractive options, mainly due to the readily availability from natural sources, similarities with the extracellular matrix (ECM), chemical versatility, good biological performances, and low processing cost. The present paper intends to overview a wide range of natural-based polymeric systems (both natural polysaccharidic systems and natural protein-origin systems) that found applications for drug delivery in tissue engineering field.

Keywords: Natural polymers, Polysaccharides, Proteins, Drug delivery, Tissue engineering.

Emerging Trends of Natural-Based Polymeric Systems for Drug Delivery


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non-communicable health problems, as non-communicable diseases need special attention in the developing countries. The point is the conditions that are currently considered as tissue engineering targets are more prevalent in the developing countries [2-3]. For exam-ple, 80% of the world mortalities due to chronic diseases and 90% of the world mortalities following traumatic injuries occur in the developing countries [2-3]. Tissue engineering is a newly engineering field using various biomaterials, cells and bioactive molecules alone or in combination to induce differentiation signals into surgi-cally transplanted formats and proliferation towards desired tissue regeneration in the damaged or diseased site [4-5]. Such strate-gies allow for developing scaffolds using various biomaterials that can be implanted with in the patient body to induce desired tissue regeneration in the damaged or diseas-ed site. Scaffolds are central components of various tissue engineering strategies, because they provide an architectural context in which extracellular matrix, cell-cell and growth factor interactions combine to generate regenerative niche [6]. The most emerging strategy for tissue engineering is relying on producing scaffolds with an informational function, e.g., material containing growth factors, proteins etc., which facilitates cell attachment, proliferation and differentiation that is far better than non-informational materials [7]. Therefore, the strategy to mimic matrix and provide necessary inform-ations like signaling for cell attachment, proliferation and differentiation to meet the requirement of dynamic reciprocity for tissue engineering. This justifies the importance of drug delivery in tissue engineering. But, there is a significant challenge in the design and development of scaffolds that possesses both highly porous structure and the ability to control the release kinetics of drugs over the period of tissue regeneration. The design and selection of a biomaterial is a key step in the development of scaffolds for tissue engineering. Generally, the ideal bio-materials used for this purpose must be safe,

non-toxic, possess acceptable biocompatibili-ty promoting favorable cellular interactions and tissue development, while possessing adequate mechanical and physical properties. They should have sufficient biodegradability and bioresorbability to support the regen-eration of new healthy tissues without any inflammation. A variety of biomaterials have been investigated and proposed to be used in the development of scaffolds, namely biode-gradable polymers of both natural and synthetic origin, and inorganic biomaterials [7-12]. Among them, natural-based polymers offer the advantage of being similar to biological macromolecules. Owing to their similarity with the extracellular ECM, natural polymers may also avoid the stimulation of chronic inflammation or immunological reactions and toxicity, often detected with synthetic polymers [13]. The degradation of natural polymers into physiological metaboli-tes makes them excellent candidates for a wide range of applications in biomedical field including drug delivery. At present, the socio-economic condition of the modern world has elevated the interest in using natural-origin biomaterials for various biomedical applications. Environmental con-cerns are also playing a considerable role, contributing to the growing interest in natural polymers due to their readily availability, acceptable biocompatibility, good biodegra-dability, low toxicity, low processing and disposal cost. The largest amount of natural polymers is still extracted from plant [14-16] and animal sources [17] and from algae [18]. Nowadays with the advancement of biotech-nology, natural polymers can be obtained by the fermentation of microorganisms or prod-uced in vitro by enzymatic process [10,13]. Various natural polymers are generally either polysaccharides or protein-derived. In this review, different natural polymer-based systems that have been widely proposed as scaffold materials to be used for drug delivery in tissue engineering applications will be overviewed.

M.S. Hasnain et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 1-13


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2. Polysaccharidic Polymeric Systems Used in Tissue Engineering Applications Starch. One of the most promising natural polysaccharide is called starch which is widely used in various biomedical applica-tions due to its inherent biodegradability overwhelming abundance and renewability. It is a mixture of glycans which is synthesized by plant and deposited in the chloroplast as their food reserve. It is stored in the form of insoluble granules which is composed of - amylase (20-30%) and amylopectin (70-80%) (Figure 1) [15]. Starch by itself is extremely difficult to process when used without additional plasticizer. In most cases, the semi-crystalline native starch structure is either destroyed or recognized, or both [19]. Water can be used as usual plasticizer in starch processing and physical properties of starch are greatly influenced with the presence of water amount. Also, use of other plasticizers like low molecular weight alco-hol renders starch more processable [19]. Additionally, blending of one or more chemically and physically dissimilar natural or synthetic polymers has shown potential to overcome these starch processing difficulties [20]. Starch has also been extensively modified chemically by oxidation [21] or grafting of acryl reactive groups [22].

Figure 1. Structure of starch.

Recently, scientists and researchers have been focused on making various novel starch-based scaffolds and microparticles for delivery of drugs like non-steriodal anti-inflammatory agents, corticosteroids etc, for bone tissue engineering applications [23-24]. Starch-based scaffolds for the purposes are generally produced by melt-based [25] and rapid prototyping [26] techniques.

Cellulose and Its Derivatives. Cellulose and its derivatives is often referred as the most abundant natural polymer in the world and therefore, it is readily available and has a low cost. Cellulose is produced by plants (cotton, wood, straw etc) as well as by microorgan-isms (bacterial cellulose, e.g. Acetobacter xylinum). It is the - (1→4) polymer of anlydroglucose (Figure 2). The biocompati-bility of cellulose is well established [27]. Cellulose is poorly biodegradable in vivo, but it can make hydrolysable by changing its higher order structure [28]. Several investi-gations have done on the use of cellulose for bone and cartilage tissue regenerations [29-30]. But, the incorporation of various bioact-ive molecules like antibiotics, growth factors, proteins etc., into this material displays desirable enhancement of various tissue regenerations.

Figure 2. Structure of cellulose.

To fabricate improved cellulose-based scaffolds for tissue engineering, several modifications have done to produce cellulose derivatives like cellulose phosphate, cellulose sulphate, cellulose acetate etc. The applica-bility of cellulose phosphate as a biomaterial for orthopedic applications was investigated to improve osseointegration of cellulose. Due to its capability of binding with various growth factors, cellulose phosphate can be used as a promising alternative biomaterial able to promoting adequate tissue regener-ation and healing response. The in vitro biocompatibility studies in cultured bone cells also showed that cellulose phosphate is non-cytotoxic, independently of the phos-phate content. However, cellulose phosphate promoted poor rates of bone cells attachment, proliferation and differentiation, which were



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attributed to the negative charge associated to the high hydrophilicity of the cellulose derivative [31]. The animal implantation studies in rabbits revealed the in vivo biocompatibility of both phosphorylated and unphosphorylated cellulose, as well as their osteoconductive properties [31]. The in vivo femoral implantation of regenerated cellulose revealed their biocompatibility, but a complete osseointegration could not be observed. Phosphorylation was therefore envisagated as the means to enhance cellulose bioactivity. The in vitro studies showed that the regenerated cellulose promotes bone cell attachment and prolife-ration; but does not mineralize in acellular simulated physiological conditions. Cellulose sponge has been also evaluated in rat femur for its permissibility in the field of bone tissue regeneration and it was found to need more time to regenerate new bone tissues than the control [32-33]. Cellulose viscous sponges have been proposed as connective tissue regeneration matrices [34]. Cellulose acetate scaffolds have also been proposed for cardiac tissue engineering applications. Chitosan. The fully or partially deacetylated form of chitin is called chitosan. It is a linear copolymer of glucosamine and N-acetyl glucosamine in a - (1→4) linkage (Figure 3), usually obtained by N- deacetylation of chitin under alkaline condition. The degree of N-acetylation together with the molecular weight are the most important parameter for its characterization and is a structural parameter influencing charge density, solubi-lity, and crystallinity, including propensity to enzymatic degradation [35]. Even, with higher degree of actylation leads to faster biodegradation rates [36]. It has been proved to be biologically renewable, biodegradable, biocompatible, bioresorbable, non-antigenic, non-toxic and cell-responsive [37]. Again, the N-acetyl glucosamine present in the structure of chitosan attracts polymorpho-nuclear leucocytes, including the release of cytokines, which favor the histoarchitectural organization of connective tissues [38]. In addition, chitosan has hydroxyl and amino groups which can be modified chemically

providing a high chemical versatility. Chitosan is also a bioadhesive polymer and the adhesive properties of chitosan in a swollen state have been shown to persist well during repeated contacts of chitosan and the substrate [39]. Due to its versatile properties as attracted much attention in tissue engineering and drug delivery fields with a wide variety of applications ranging from bone, skin, cartilages etc.

Figure 3. Structure of chitin (a) and chitosan (b). Porous chitosan matrix may be suggested as a potential candidate for bone regeneration due to having its proper biological and physical properties. The biological activity of chitosan for bone generation has already reported [37]. However, chitosan has some limitations in inducing rapid bone tissue regeneration at initial stages. New bone tissue formation after implanting these matrices occurs over a very long period. This may be several months or years [37]. Incorporation of active mole-cules such as growth factors has been used as a strategy to induce improved bone tissue regeneration rapidly. Numerous bone filling materials have been developed in which chitosan is used in combination with other biomaterials, essentially as a binding agent, or associated to various biologically signaling and antimicrobial molecules [10,40]. In cartilage tissue engineering, use of chitosan is highly beneficial due to having structural similarity with glycolsamino-glycans, which is found in extracellular matrices as in native articular cartilage and are very important in playing a key role in modulating chondrocytes morphology, differ-entiation and function [10]. Chitosan has



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been extensively investigated as a wound and burn dressing material due to its easy applicability, good oxygen permeability, high water absorptivity, haemostatic property and ability to induce interleukin-8 from fibrino-blasts, which is involved in the migration of fibrinoblasts and endothelial cells [41]. Additionally, it has fungistatic and bacterio-static properties, which are particularly important for wound management. Chitosan has been found to have an acceleratory effect on tissue engineering process due to its polycationic nature. This enhances the fav-orable cell-biomaterial interaction. Alginates. In drug delivery field alginates are one of the naturally occurring polysacchar-idic polymer which is studied frequently. They are abundant in nature and are found as structural components of brown marine algae i.e. Ascophullum nodosum, Laminaria hyper-borean, Macrocystis pyrifera [39] and bacterias i.e. Pseudomonas mendocina, Azotobacter vinelandii [18]. Alginates are belonging to a family of linear copolymers composed of β-D- mannuronic acid mono-mers (M–block), regions of ∞-L- guluronic acid residues (G–block), and regions of interspersed M and G units (Figure 4). Mostly, alginates are frequently soluble in water which forms solution having

Figure 4. Structure of alginic acid.

considerable viscosity due to its rheological properties, it is used as thickening as well as gelling agent, as a colloidal stabilizer and as a blood expanders [41]. Alginates undergoes ionic gelation in aqueous solution in the presence of divalent and trivalent cations like Ca2+, Al3+ etc and the gelation depends on the ion binding [17]. In most cases, sodium

alginate is used to crosslink with these divalent cations. The crosslinking process can be carried out under mild conditions at normal room temperature excluding any use of organic solvents, and hydrogels of different shapes can be prepared. One of the drawbacks of alginate-based hydrogels is that the degradation occurs via uncontrollable degradation kinetics and hydrogels dissolve in a unpredictable manner following the loss of divalent cations releasing high and low molecular weight alginate units [43]. Several therapeutic agents including antibiotics, enzymes, growth factors, DNA, etc have already been successfully incorporated in alginate hydrogels [42]. Moreover, alginate-based hydrogels have been extensively studied for bone and cartilage tissue engineering applications as scaffolds and vehicles for biologically active molecules [44-45]. The use of poorly water soluble salts like calcium carbonate (CaCO3), calcium sulphate (CaSO4) influences gelation rate and consequently, mechanical properties [46]. When tricalcium phosphate (TCP) is used to promote gelation, it additionally promotes osteoconduction that can facilitate cell-attachment [47]. Such injectable gels were also mixed with insulin like growth factor, resulting in a seven fold increase in prolife-ration of osteoblast-like cells [47]. The enhanced adhesion of osteoblast-like cells to calcium phosphate-alginate microspheres of different compositions loaded with the recombinant enzyme, glucocerebrocidase in comparison with their polymeric counterparts has been investigated [48]. The ceramic-to-polymer ratio strongly influenced the ability of cells to adhere and spread on microspheres surface. Due to its versatile bio-character-istics, alginate-based scaffolds are by far one of natural polymers applied in various tissue engineering applications even consider growth factor delivery or cell encapsulation. Dextran. A high molecular weight polymer of D-glucose is called dextran. It is produced by bacterial sources like Leuconostoc mesent-eroids. Dextran is a branched polysaccharide consisting of ∞ (1→ 6) - linked D- glucose residues with some degree of branching via ∞



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(1→3) linkages. It is readily available in a wide range of molecular weight along with several derivatives. Both the branching and molecular weight distribution affect its physicochemical properties [49]. The biode-gradability and biocompatibility of dextran make it suitable for wide range of biomedical applications. Additionally, it has been shown to be a bone healing promoter and also has capacity of dermal and subcutaneous augm-entation. It has wide applications in drug delivery [49].

Hyaluronic Acid. Hyaluronic Acid also known as hyaluronan or hyaluronate, is a major macromolecular component of the intercellular matrix in most connective tissues such as articular cartilage, vitreous of the human eye and synovial fluid (the fluid that lubricates joints) [50]. Hyaluronic acid is a linear polysaccharide that chemically composed of alternating disaccharide units of ∞-1, 4-D-glucuronic acid and β-1, 3-N-acetyl-D-glucosamine, linked by β (1→3) bonds (Figure 5).

Figure 5. Structure of hyaluronic acid.

Hyaluronan is water soluble and can form hydrogels by covalent and photocrosslinking with hydrazide derivatives, by esterification and annealing [51]. Hyaluronan and its associated networks possesses many physio-logical roles that include tissue and matrix water regulation, structural and space filling properties, lubrication, and a number of macromolecular functions [51]. Especially for its enhanced viscoelastic properties, hyaluronan works as lubricant and shock absorber in synovial fluid [52]. It has also

capacity to act as a scavenger for free radicals in wound sites, there by modulating inflam-mation and can interact with a variety of biomolecules. Hyaluronic acid possesses several important properties that make it an candidate for wound dressing applications [41]. Again, it has bacteriostatic property. Hyaluronan has been widely studied for drug and gene delivery. Hyaluronic acid can be recognized by receptors on a variety of cells associated with tissue repair. Hyaluronic acid for drug delivery in tissue engineering applications has been focused on cartilage, bone and osteochondral applications, most likely due to the fact that is macromolecular component of the extracellular matrix [10]. Carrageenans. A family of sulphated poly-saccharides extracted from red marine algae are called carrageenans. They are linear polymers consisting of chains of (1→3) - linked β-D-galactose and (1→4)-linked ∞-D-galactose units, which are variously substi-tuted and modified to the 3, 6-anhydro derivative depending on the source and extraction conditions [53]. Three major types of carrageenan are recognized on the basis of their patterns of sulphate esterification: kappa (κ), iota (ι), and lambda (λ). All these carrageenans are highly flexible molecules, which, at higher concentrations, wind around each other to form double helical structure. Carrageenans have been used in the field of drug delivery [54-55], which creates the potential for their use as drug delivery systems in various tissue engineering applications.

Gellan Gum. A high molecular weight bacterial exopolysaccharide produced by Pseudomonas elodea is called gellan gum. It is a linear anionic heteropolysaccharide composed of the tetrasaccharide (1→ 4)- L- rhamnose - ∞ (1→3)- D-glucose- β (1→4)- D-glucuronic acid - β (1→4)- D-glucose as a repeating unit. High acyl gellan gum gives soft, elastic, transparent gels at polymer concentrations higher than 0.2 %. But, low acyl gellan gum can form hard, brittle and non-elastic gels [13]. Gellan gum has been studied for drug delivery applications, with



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both as adjuvant and as vehicle for drug delivery [56-57]. Suri and Banerjee has first explained its use for tissue engineering applications, where they have used a gellan gum gel as a substitute of the vitreous of the eye, and its properties were comparable to the commonly used material (silicone) [58]. Its properties is similar to other materials already studied in the field of tissue engineering, which make this natural polymer a suitable candidate for both drug delivery and cell encapsulation in various tissue engineering applications. Table 1 intends to summarize some relevant applications of natural poly-saccharidic polymer-based matrices/scaffolds for drug delivery in various tissue engineer-ing fields. 3. Protein-Origin Polymeric Systems Used in Tissue Engineering Applications Collagen. Collagen as a natural polymer, is increasingly being used as a device material in tissue engineering and repair. It is the major protein component of the extracellular matrix providing the support to connective tissues such as skin, tendons, bones, cartilage, blood vessels and ligaments [75-76]. There are various types of collagen have been identified (i.e. approx 27 types). Among them, collagen type I is the most abundant collagen, which is frequently investigated for various biomedical applications. Collagen possesses good biocompatibility, low anti-genicity, and high mechanical strength. Collagen can crosslinked and tailored for its degradation and water uptake properties. There are various factors which influenced the degradability of collagen, like the penet-ration of cells into the structure causes, and as well as the fact that several other non-specific proteinase are able to digest collagen besides collagenase and gelatinase [10]. Various collagen-based scaffolds for tissue regeneration are already investigated [10,76]. It is used in bioengineered artificial skin production, which is approved by FDA in 1998. It is also used in bone graft. But, incorporation of various drug and signaling molecules can improve the tissue regener-

ation in the desired and damaged site. Recently, there is a growing interest in development of various collagen-based mat-rices and scaffolds for drug molecule, cell, gene delivery in different tissue engineering applications. Gelatin. Gelatin is a denatured protein which is obtained by acid and alkaline processing of collagen and is commonly used in various pharmaceutical and biomedical applications [77]. It has also biodegradability and bio-compatibility in physiological environment. It has relatively low antigenicity, because it is denatured in contrast to collagen. The later one has antigenicity due to its animal origin. Gelatin contains a large number of glycine, proline and 4-hydroxy proline residue. Two different types of gelatin can be produced based on the method in which collagen is pretreated, prior to the process of extraction [78]. The different processing conditions of gelatin allows for flexibility in terms of enabling polyion complexation of a gelation carrier with either positively or negatively charged biomolecules. If the biomolecules to be released is acidic, basic gelatin with an isoelectric point of [9] is preferable as a matrix, while acidic gelatin with isoelectic point of [5] should be applicable to the control release of a basic protein. Both gelatins are insoluble in water to prepare a hydrogel by crosslinking with water soluble carbodimides and glutaraldehyde [77]. Gela-tin exhibits essentially the same common properties typical polymeric substances, which is not the case with native collagen. Due to its processing property and the possibility of polyion to complex formation, it has been widely used as matrices and scaffolds for drug delivery in various tissue engineering applications. Incorporation of drug molecules, growth factors, and other cell-adhesion proteins and peptides within the gelatin-based system is also a strategy to be implemented to achieve a successful tissue engineering approach [79]. Fibrin. From fibrinogen, fibrin is isolated by providing an immunocompatible carrier for delivery of active biomolecules; it can be



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autologously harvested from the patient [80]. Fibrin is frequently investigated as a sub-stitute for cell adhesion, spreading migration and proliferation because it contains sites for cell binding [81]. Polymerized fibrin is a major component of blood clots and plays a vital role in the subsequent wound healing response [82]. Fibrin glue is a biological adhesive due to which it is also used in

surgery like abdominal, thoracic, vascular etc. This is because of its haemostatic, chemostatic and mitogenic properties. It is used as a cell carrier to various cell types like keratinocytes, tracheal epithelium cells, urothelium cells, murine embryonic cells and to encapsulate chondrocytes for cartilage tissue engineering [83-85].

Table 1. Natural polysaccharidic matrices/scaffolds for drug delivery in tissue engineering. Natural polysaccharidic Drugs Animal Tissue engineering matrices/scaffolds model applications Starch-based porous scaffolds [59] NSAIDs - Bone Starch-based microparticles [23] NSAIDs - Bone Starch-based microparticles [60] Corticosteroids - Bone Cellulose hollow fibers [61] Fibrinectin - Not defined Chitosan hydrogel [62] FGF-2 Rabbit myocardial Vascularization Infarction Chitosan hydrogel [63] EGF Rat burn wounds Skin Chitosan hydrogel [64] FGF-2 Mice full-thickness Skin skin incision Chitosan granules in a PGDF Rat femur defect Bone TCP/chitosan hydrogel [65]

Alginate-chitosan fibers [66] Dexamethasone, - Not defined PDGF-BB Alginate beads [67] TGF-β Rabbit knee Cartilage Osteochondral defects Alginate beads [68] BDNF Rat sciatic nerve Peripheral nerve regeneration Alginate beads [69] bFGF, VEGF, Rat myocardial Vascularization EFG infarction Alginate hydrogel [70] bFGF, VEGF Nude mice Vascularization subcutaneous implantation Hyaluronic acid gel [71] bFGF Nude mice Vascularization subcutaneous implantation Hyaluronan-alginate scaffold [72] bFGF - Not defined Carboxymethyl-Dextran Lysozyme - Not defined hydrogel membranes [73]

Gellan gum (Gelrite®) [74] Antibiotic Rabbit bacterial Ophthalmology conjunctivitis Abbreviations: NSAIDs: Non-steroidal anti-inflammatory drugs; PDGF-BB: Recombinant human platelet-derived growth factor-BB; TGF-β: Transforming growth factor-β; BDNF: Brain-derived neutrophic factor; bFGF: Basic fibrinoblast growth factor; VEGF: Vascular endothelial growth factor; EFG: Epidermal growth factor; FGF-2: Fibrinoblast growth factor-2; PGDF: Platelet-derived growth factor



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Table 2. Protein-origin polymeric systems used for drug delivery in tissue engineering. Protein-origin Drugs Animal Tissue engineering matrices/scaffolds model applications Collagen/hydroxylapatite [86] NGF Calvaria defects Bone Collagen gel [87] VEGF Corioallantoic Vascularization membrane Gelatin Sponge [88] BMP-2 Tracheal cartilage Cartilage rings in canine cervix Gelatin-siloxane [89] Gentamicin - Bone sulfate Fibrin gel [90] ngl BMP-2 Critical size defects Bone in rat calvarium and inter-carpal fusion in dogs Abbreviations: NGF: Nerve growth factor; VEGF: Vascular endothelial growth factor; EFG: Epidermal growth factor; BMP-2: Bone morphogenetic protein-2; nglBMP-2: Non-glycosylated form of bone morphogenic protein-2.

Table 2 intends to summarize some relevant applications of natural protein-origin poly-mer-based matrices/scaffolds for drug deliv-ery in various tissue engineering fields. 4. Conclusions Natural-based polymeric systems for drug delivery and tissue engineering applications have received a considerable interest. How-ever, the combination of both applications into a single material has proven to be very challenging. In this field, a great deal of effort has been put in to fabricate different formulations of scaffolds based on natural polymers. Many attempts have been made

to produce various smart systems using various natural polymers that could be used in tissue engineering applications, including fabrication of scaffolds or hydrogels that can deliver relevant drug molecules like antibiotics, proteins, growth factors etc. Although, natural polymers present some limitations such as difficulties in controlling the variability from batch to batch, mech-anical properties or limited processability. But, their degradability, biocompatibility, low cost and availability, similarity with ECM and intrinsic cellular interactions makes them attractive candidates for various biomedical applications, in particular as drug delivery systems for tissue engineering applications as described in this review.

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Research Article

Nano-Sized Titanium Dioxides as Photo-Catalysts in Degradation of Polyethylene and Polypropylene Packagings

T. Manangan1,2*, S. Shawaphun1,2, D. Sangsansiri1, J. Changcharoen1, S. Wacharawichanant3

1Department of Industrial Chemistry, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand.

2Research Center of Nano Industries and Bio-plastics, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand.

3Department of Chemical Engineering, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom 73000, Thailand.


Abstract

In present, packaging plastics such as PE and PP have become a major environmental problem. While production of biodegradable plastics is still expensive, various photo-catalytic additives especially titanium dioxide have been used as pro-oxidants in order to make conventional packaging plastics degradable after working period. Various particle-sized titanium dioxides were thermally blended with PE and PP and casted into 80-micron thick films. During the processing period, nano-sized TiO2 significantly induced auto-oxidation of the PE films only. The carbonyl formation in PP films showed the opposite trend possibly due to other mechanical pathways. The films then were exposed under 254nm and 366nm UV light mimicking solar light profile. In most cases, TiO2 catalyzed photo-degradation occurred under the shortwave UV-254nm irradiation several folds higher than under long wave UV irradiation. The carbonyl index of the nano-sized TiO2 (1%wt) blended PE films increased continuously over irradiation period and their tensile strength reduced to 35-38% after 28 days up to 42 days before total ruptured. The films have also lost weight about 11-15 % in 14 days. The nano-sized TiO2 blended PP films showed a dramatic increasing of carbonyl index in the first few days and then continuously dropped as they become fragmented with in 2 weeks. This also caused PP films to lose weight by 22% in 42 days. This study also suggested that titanium dioxide nano-sized particle showed favorable activity and results over the commercial micron-sized.

Keywords: Photo-catalysts, Degradation, Plastic packaging, Titanium dioxide.

T. Manangan et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 14-20


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1. Introduction

Polyethylene and polypropylene are the most popular plastics in packaging industries due to their useful mechanical properties and physical properties for foods and household products packaging. However, these plastics take several years to decompose. Therefore, gigantic amount of the petroleum based plastic packaging has been disposed into the environment and caused serious natural resource contamination every year [1]. Many countries have urged to apply various regulations, policies and managements to overcome these problems e.g. recycle reuse and reduce protocol, yet it is far from success. Moreover, bio-plastics and bio-degradable plastics have also become worldwide main research area in order to solve such waste problems [2]. However, large scale production and marketing of such plastics are still very expensive. So many plastic producers have developed oxo-degradable plastics from the conventional plastics and made them degradable after used. In this communication, the oxo-degradability has been introduced in situ using photo-catalytic additives that can also act as the thermal oxidative catalyst during the thermal plastic film processing. Typic-ally, metal oxides or metal salts such as Mn2+/Mn3+, Fe3+, Zn2+, Zr2+, Ce2+ and Ti4+ have been used in such purpose [3-6]. In our previous work, thermal oxidative activity of TiO2 compared with other metal oxides was preliminarily studied by monitoring carbonyl index of the blended plastic films and hydrocarbon solvents. Only ZnO2 and TiO2 were found to give good activity. In addition, TiO2 showed approximately 2 folds higher activity than ZnO2 [7]. Due to its superb characteristics such as inexpensiveness, non-toxicity, stability and highly photo-active-ness, TiO2 has become the excellent choice for photo-catalyst in order to make the conventional packaging plastics such as PE, PP and PS become degradable after working period, especially for food contacting plastics [8-9]. Since, TiO2 can also be thermal catalyst during the processing period and induce the auto-oxidation of plastics. It is

often referred as a pro-oxidant or sometimes called pro-degradants. Plus, several groups of the nano-sized TiO2 have been synthesized and proven to give superior catalytic behavior over the micro-sized TiO2 in many reactions. Hence, it is possible to be used effectively in the plastic degradation.

2. Theory

Typically, a natural pathway of auto-oxidative degradation of polyethylene and polypropylene is normally initiated by light (photo-oxidative degradation) or heat (thermo -oxidative degradation). This step usually is the slowest process and becomes the rate determining step, because plastics have to combine with oxygen in the air and react to give the unstable peroxides which can then decompose to carbonyl and free radical inter-mediates e.g. alkyl radicals or hydroxyl radicals [10]. Moreover, the carbonyl inter-mediates can be photolyzed to more free radicals [11]. The radicals then undergo propagation step in the chain reaction of PE and PP degradation and give more of oxygen-ated carbon skeletons in the plastics in form of hydroxyls, aldehydes, ketones and carboxylic acids which then can be further biodegraded at low molecular weight ultimat-ely resulting carbon dioxide and water [12].

The objective of this research is to accelerate the rate determining step by using TiO2 pro-oxidants which can generate carbonyl intermediates during the thermoplastic pro-cessing step and catalyze degradation of plastics under solar light exposure after used and casted away. Moreover, the thermal oxidative and photolytic reactivity of nano-sized TiO2 particle was also investigated as well as crystalline types e.g. anatase and rutile. The TiO2 with various particle sizes, crystalline types, grades, and additive concentrations were blended into LDPE and PP, casted into a thin film, irradiated under ultraviolet light at 254nm and 366 nm. Tensile strength, modulus and elongation at break of the films and carbonyl index were measured thoroughly to understand their catalytic degradation pathways.

Nano-Sized Titanium Dioxides as Photo-Catalysts in Degradation


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3. Materials and Methods

Virgin grade LDPE (IRPC) and PP (IRPC) were used for the preparation of films. Commercial analytical grade TiO2 (predo-minantly rutile, referred as TiO2-com), nano-sized rutile TiO2 (rutile, particle size < 100 nm, SSA > 14 m2/g), nano-sized anatase TiO2 (anatase, particle size < 25 nm, SSA 200-220 m2/g), benzene (HPLC grade) and hexanes (HPLC grade) were purchased from Sigma Aldrich®. The micron-sized TiO2 (rutile, particle size < 63 µm) was prepared from TiO2-com by particle size sieving at 230 Mesh and referred as TiO2-63µm. Most catalysts were oven dried for 24 hours and kept in desiccators before use.

Catalytic Thermal Oxidation of PE and PP Films during Processing. The powders of TiO2-com, TiO2-63µm, nano-sized rutile TiO2 and nano-sized anatase TiO2 were blended into both LDPE and PP at 0%, 1%, 2%, 3%, and 5% w/w concentrations using a Twin-Screw Extruder (TSE 16 TC; Intro enterprise Co., Ltd.). The heating profile was set as followed: feed zone 120oC, compression zone 140oC and metering zone 160oC. Screw speed was set at 40 rpm. Then the obtained plastics were casted to 80-micron thick films using a Chill Roll Cast Film Machine (LE 25-30/C; Labtec Engineering Co., Ltd.) with screw speed at 50 rpm and six heating zones set at 170 oC, 190

oC, 200 oC, 210 oC, 210 oC and 210oC. Tensile strength of the films was measured using a Universal testing machine (Tensile H5K-T; Calserve Thailand Co., Ltd) to compare with the virgin film. The film carbonyl index (CI) was measured via ATR-IR spectroscopy using a Perkin Elmer spectrum 2000 FTIR spectrometer and calculated by the ratio of the peak area between 1640–1840 cm-1 (C=O stretching band) and the peak area between 1350–1470 cm-1 (C-H bending band).

Catalytic Photo-Oxidative Degradation of PE and PP Films under UV Lights. The obtained plastic films containing various types of TiO2 which possess about the same carbonyl index

were cut to 1 x 5 cm2 size and exposed under a 20-watt shortwave UV lamp (average wavelength at 254 nm) and a 20-watt long wave UV lamp (average wavelength at 366 nm) at 30 cm in distance. The plastic films then were taken out daily or weekly to measure tensile strength, elongation at break, modulus, carbonyl index and % weight loss to determine their degree of degradation.

4. Results and Discussion

Thermal Oxidation of PE Films. The effect of particle size in thermal oxidative degradation of LDPE films, only TiO2 with rutile crystalline were used as catalysts in this investigation. After the powders of TiO2-com, TiO2-63µm and the nano-sized rutile TiO2 were blended into LDPE at various concentrations and then casted to 80-micron thick films, it was found that the film without TiO2 showed very low carbonyl index at 0.023. This indicated that the heat as high as 210 oC during plastic film processing did not induce auto-oxidation. However, the LDPE film blended with TiO2-com 1%w/w via the same process showed carbonyl index at 0.535 similarly to the film blended with the nano-sized rutile TiO2 1%w/w. It is important to note that the size-sieved TiO2-63µm gave slightly higher catalytic oxidative activity than TiO2-com at higher concentrations as shown in Figure 1. The particle size effect became so clear when the nano-sized rutile TiO2 was blended with LDPE under the same

Figure 1. Effect of TiO2 particle size, structure and concentration on catalytic thermal oxidation of LDPE films during processing.



17

processing condition at high concentration 3% and 5% w/w. The nano-sized TiO2 provided thermal oxidative activity by 2 - 2.5 folds over TiO2-com. This is probably due to dispersion effect only. Because the LDPE films blended with 1% and 2% w/w of most rutile TiO2 catalysts have nearly the same carbonyl index. Though, the nano-sized rutile catalyst has particle size less than 100 nm and a specific surface area more than 14 m2/g.

In addition, the nano-sized anatase TiO2 blended LDPE film under the same processing condition showed significantly higher carbonyl index than the film blended with rutile crystalline in all catalyst concentrations. The nano-sized anatase TiO2 not only processes particle size less than 25 nm, but also process specific surface area of 200-220 m2/g or about 15 times greater than the nano-sized rutile specific surface area. Hence, its catalytic oxidative activity has been superb.

Figure 2. Tensile strength of LDPE films containing rutile TiO2 at various particle sizes after thermal oxidative processing.

Similar to carbonyl index, TiO2-com and TiO2-63µm gave nearly identical results on the LDPE film tensile strength during the film processing period in all additive concentrations as shown in Figure 2. However, most of the blended films have slightly lower tensile strength than pure LDPE. This suggested that these LDPE films still possess normal mechanical properties for packaging uses. The LDPE film blended with both nano-sized TiO2 also showed lowering of the film tensile strength as the additive

concentration increased. This is possibly due to the degradation via scission mechanism already occurred in this high temperature film processing period. Again, nano-sized anatase catalyst showed better reactivity than rutile catalysts in all concentrations.

Thermal Oxidation of PP Films. The effect of particle size in thermal oxidative degradation of PP films was carried out exactly the same as in LDPE film. After the powders of the TiO2-com, TiO2-63µm and the nano-sized TiO2 with rutile crystalline were blended into PP at various concentrations and film casted, it was found that the pure PP film without TiO2 also showed very low carbonyl index at 0.013 indicating that the heat as high as 210 oC during plastic film processing did not induce auto-oxidation. However, the film blended with TiO2-63µm showed carbonyl index about 0.80-1.40 similar to the film blended with TiO2-com as shown in Figure 3.

Figure 3. Effect of TiO2 particle size on thermal oxidation of PP films during processing.

Figure 4. Tensile strength of PP films containing various sized rutile TiO2 and nano-sized anatase TiO2 after processing.



18

Surprisingly, the nano-sized TiO2 both anatase and rutile showed lower oxidative activity in PP film than the commercial micron-sized rutile especially at low catalyst content. Furthermore, the tensile strength also has become even greater at high catalyst content as shown in Figure 4. This is possibly due to some recombination of the generated radicals instead of scission or oxidation [9].

Catalytic Photo-Oxidative Degradation of PE and PP Films under UV Lights. In order to minimize the difference of the carbonyl content, the blended LDPE and PP films containing 1% w/w of various TiO2 which process about the same initial carbonyl index were cut to 1 x 5 cm size and exposed under a 20-watt shortwave UV lamp (254 nm) and a 20-watt long wave UV lamp (366 nm) at 30 cm in distance. Moreover, the films were also exposed under both lamps to mimic the solar light profile. The plastic films then were taken out daily to measure tensile strength, carbonyl index and % weight loss to determine their degree of degradation. It was found that most of these catalysts were highly active only under 254-nm ultraviolet irradia-tion. The results under dual lamp condition gave almost identical to the 254-nm ultra-violet irradiation alone (results not shown here). Hence, most of the photo-oxidative degradation was done under a 20-watt short-wave UV lamp (254 nm) only.

Most LDPE films blended with TiO2 slowly increased their carbonyl index in the first 24 hour and then dramatically increased later after. The carbonyl index kept on rising until the film surface became brittle and ruptured. The cloudy films were usually observed after 72 hours and the carbonyl index tended to drop due to ATR-IR scattering interference as shown in Figure 5. These results fitted perfectly with the tensile strength profile as shown in Figure 6. Most of the film tensile strength appeared to remain constant up to 48 hour of irradiation and then drop drastically right after as the film fragmentation occurred and the carbonyl index started to drop.

Figure 5. Initial effect of TiO2 (1%w/w) on carbonyl index of LDPE films under 254-nm UV irradiation.

Among these TiO2 catalysts, the nano-sized showed the highest photo-oxidative degrade-ation of LDPE films especially the nano-sized anatase form which can lower the film tensile strength by 38% in 96 hours under UV irradiation compared to that of pure LDPE under the same condition as depicted in Figure 6.

Figure 6. Initial effect of TiO2 (1%w/w) on tensile strength of LDPE films under 254-nm UV irradiation.

In the other hand, PP films blended with the nano-sized TiO2 did not show much increase of their carbonyl index, while the commercial TiO2-com and the micron-sized TiO2-63µm blended films showed similar trend as in LDPE films as shown in Figure 7. Further-more, the film tensile strength appeared to increase under UV irradiation as shown in Figure 8. The catalytic photo-degradation of these PP films seemingly underwent via a different mechanism.



19

Figure 7. Initial effect of TiO2 (1%w/w) on carbonyl index of PP films under 254-nm UV irradiation.

Figure 8. Initial effect of TiO2 (1%w/w) on tensile strength of PP films under 254-nm UV irradiation.

To further understand and predict total rupture of these LDPE and PP films under solar light irradiation, the films then were exposed under 254-nm ultraviolet for longer period. The films then were taken out weekly to determine their degree of degradation by measuring carbonyl index, tensile strength, modulus, % elongation at break and % weight loss. It was found that the carbonyl index (using FTIR, KBr) of both nano-sized TiO2 (1%wt) blended LDPE films increased continuously over irradiation period and their tensile strength reduced to 35-38% after 28 days up to 42 days before total ruptured. The films have also lost weight about 11-15 % in 14 days.

Both nano-sized TiO2 blended PP films showed a dramatic increasing of carbonyl index in the first few days and then conti-nuously dropped as they became fragmented

with in 2 weeks. This also caused PP films to lose weight by 22% in 42 days.

5. Conclusions

Titanium dioxide catalysts can be used as packaging plastic pro-oxdiants or pro-degradants which can actively catalyze thermal oxidation generating carbonyl inter-mediates during the film processing and also act as the photo-oxidative degradation catal-ysts along with the carbonyl intermediates under the UV and solar lights irradiation. During the processing period, both nano-sized TiO2 significantly induced auto-oxidation of the PE films. However, carbonyl formation in PP films suggested other mechanical pathways. After an exposure under 254nm and 366nm UV light mimicking solar light profile, in most cases, TiO2 catalyzed photo-degradation occurred under the shortwave UV-254nm irradiation several folds higher than under long wave UV irradiation. The carbonyl index of the nano-sized TiO2 (1%w/w) blended PE films increased continuously over the irradiation period and their tensile strength reduced to 35-38% after 28 days up to 42 days before total ruptured. The films have also lost weight about 11-15 % in 14 days. The nano-sized TiO2 blended PP films showed a dramatic increasing of carbonyl index in the first few days and then continuously dropped as they become fragmented with in 2 weeks. This also caused PP films to lose weight by 22% in 42 days. This study also suggested that titanium dioxide nano-sized particle showed significantly more favorable in both thermo- and photo-oxidative degradation catalytic activity and results than the commercial and micron-sized TiO2.

Acknowledgements

The authors would like to acknowledge the financial support from the Research, Devel-opment and Engineering (RD&E) Fund through National Nanotechnology Center (NANOTEC), National Science and Techno-logy Development Agency (NSTDA), Thai-



20

land (Project NN-B-22-CT5-17-51-17) and also the National Research Council of Thai-

land (NRCT).

References

[1] Guillet, J. (1995). In: D. Gilead, & G. Scott

(eds.), Degradable Polymers Principles & Applications. London: Chapman & Hall.

[2] Sudesh, K., Abe, H. & Doi, Y. (2000). Synthesis, structure and properties of poly-hydroxyalkanoates: biological polyesters. Progress in Polymer Science, 25, 1503-55.

[3] Jin, C., Christensen, P.A., Egerton, T.A., & White, J.R. (2003). Effect on anisotropy on photochemical oxidation of PE. Polymer, 44, 5969-81.

[4] Scott G., & Islam, S. (1999). Polymer-bound activators for polyolefins. Polymer Degrad-ation and Stability, 63, 61-4.

[5] Jakubowicz, I., Yarahmadi, N., & Peterson, H. (2006). Evaluation of the rate of abiotic degradation of biodegradable polyethylene in various environments. Polymer Degradation and Stability, 91, 1556-62.

[6] Jakubowicz, I. (2003) Evaluation of degrada-bility of biodegradable polyethylene (PE). Polymer Degradation and Stability, 80, 39-43.

[7] Shawaphun, S., Manangan, T., & Wachara-vichanant, S. (2010). Thermo- and photo-degradation of LDPE and PP films using metal oxides as catalysts, Advanced Materials Research, 93-94, 505-9.

[8] Qin, H., Zhao, C., Zhang, S., Chen, G., & Yang, M. (2003). Photo-oxidative degrada-tion of polyethylene/montmorillonite nano-composite. Polymer Degradation and Stabi-lity, 81, 497-500.

[9] Zhao, X., Li, Z., Chen, Y., Shi, L., & Zhu, Y. (2007). Solid-phase photo-catalytic degradat-ion of polyethylene plastic under UV and solar light irradiation. Journal of Molecular Catalysis A: Chemical, 268, 101-6.

[10] Gijsman, P., Meijers, G., &Vitarelli, G. (1999). Comparison of the UV-degradation chemistry of polypropylene, polyethylene, polyamide 6 and polybutylene terephthalate. Polymer Degradation and Stability, 65, 433-41.

[11] Chiellini, E., Corti, A., D’Antone, S. & Baciu, R. (2006). Oxo-biodegradable carbon backbone polymers- Oxidative degradation of polyethylene under accelerated test condit-ions. Polymer Degradation and Stability, 91, 2739-47.

[12] Haines, J.R., & Alexander, M. (1975). Microbial degradation of high-molecular weight alkanes. Applied Microbiology, 28, 1084-5.



SCIENCE JOURNAL Ubon Ratchathani University http://scjubu.sci.ubu.ac.th

Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 21-26

Research Article

Effect of Zinc Oxide on the Morphology and Mechanical Properties of Poly(Styrene-co-Acrylonitrile)/Poly(Methyl

Methacrylate)/Zinc Oxide Composites

S. Wacharawichanant*, N. Thongbunyoung, P. Churdchoo, T. Sookjai

Department of Chemical Engineering, Faculty of Engineering and Industrial Technology, Silpakorn University, Nakhon Pathom 73000, Thailand.


1. Introduction

There is currently great interest in the intro-duction of inorganic particles to organic polymers because organic–inorganic compos-ites offer an effective way to improve the

physical properties of conventional polymers such as mechanical properties, thermal stabi-lity, flame retardancy, electrical properties, and chemical reagent resistant [1-6]. Polymer composites are widely used in applications such as transportation, construction, electro-nics and consumer products. The properties of particle-reinforced polymer composites are strongly influenced by the dimensions and microstructure of the dispersed phase [7].

Abstract

The effect of zinc oxide (ZnO) on the morphology and mechanical properties of poly(styrene-co-acrylonitrile) (SAN) and poly(methyl methacrylate) (PMMA) blends was investigated. Composites of blends of SAN and PMMA (20/80 wt%) with ZnO were prepared by melt mixing in a twin screw extruder and then the composites were molded by compression molding method. The dispersion of ZnO particles in the matrix polymers was investigated using SEM. It was observed that the dispersion of ZnO particles was relatively good with low ZnO content but the aggregates of ZnO particles in a polymer matrix increased with increasing ZnO content. The mechanical test showed that the tensile strength and stress at break of SAN/PMMA blends decreased slightly with increasing ZnO content. Increasing content of ZnO up to 1.0 wt% increasing the Young’s modulus and impact strength of SAN/PMMA blends while the addition of ZnO beyond 1.0 wt% decreased the Young’s modulus and impact strength.

Keywords: Zinc oxide, Poly(styrene-co-acrylonitrile), Poly(methyl methacrylate), Morphology, Mechanical properties.

Effect of Zinc Oxide on the Morphology and Mechanical Properties of Zinc Oxide Composites


22

Poly(methyl methacrylate) (PMMA) is an optically clear amorphous thermoplastic. It is widely used as a substitute for inorganic glass, because it shows higher impact stren-gth and undergoes ductile rather than brittle fracture [8]. Copolymers are increasingly important industrially because the introduct-ion of one component may make up for shortcomings due to the other component. One representative copolymer is poly(styrene -co-acrylonitrile) (SAN), which exhibits the combined properties of the ease of processing of polystyrene and the rigidity and chemical resistance of polyacrylonitrile [9]. Zinc oxide (ZnO) particles are relatively easy to dis-perse, and compared to other particles such as aluminum oxide (Al2O3) or titanium dioxide (TiO2), ZnO particles have almost no surface water [10].

It is well known that the mixture of PMMA and SAN forms a miscible blend and exhibits lower critical solution temperature (LCST) phase-separation upon heat-treatments. When temperature changes, it is possible to obtain blends with various phase morphology (sing-le or separated) and different viscoelastic properties [11-13].

Lee et al. [11] investigated effect of clay on the morphology and properties of PMMA/-SAN/clay nanocomposites. It found that the clays were observed to be mainly located at the boundaries of PMMA and some of them in PMMA domains. As the number of pass increased, the phase-separated domain size became larger and differential scanning calorimetry (DSC) result for this nanocom-posite shows lowering in glass transition temperature and widening of glass transition region.

In this study, composites of PMMA/SAN blends (20/80 wt%) with ZnO were prepared by melt processing in a twin screw extruder and the effect of ZnO on the morphology and mechanical properties of composites was investigated. Analyses were carried out using universal tensile testing machine, Pendulum

impact tester and scanning electron micro-scope (SEM) to investigate the dispersion of ZnO and its influence on the mechanical properties of composites.

2. Theory

The incorporation of inorganic particles into polymers allows one to integrate new funct-ions inside polymer matrixes [14]. The properties of a polymer-particle composite depend on a number of parameters, such as the particle size and volume fraction, the particle-polymer interaction energies, and polymer chain length and composition. These parameters are intimately related to the properties and performance of materials [15]. The addition of fillers into polymers results in two different interactions, that is, polymer–filler interaction and filler–filler interaction. These two interactions affect the flow behavior and mechanical performance of the composites [16].


Materials. The copolymer of SAN was suppl-ied by LANXESS Co., Ltd. The glass transition temperatures (Tg) of the SAN was around 105C. The commercial grade PMMA was supplied by Diapolyacrylate Co., Ltd. and for this samples Tg is 95C. ZnO in the form of a white powder with average particle sizes of 250 nm was purchased from S.R.LAB Co., Ltd. Sample Preparation. SAN and PMMA pellets and ZnO particles were dried in an oven at 110 C for 3 hrs before melt extrusion. Composites of blends of SAN and PMMA (20/80 wt%) with ZnO were prepared by melt mixing in a twin screw extruder at temperatures in a range of 180-220 C and a screw speed of 50 rpm. After compounding, the composites were compression-molded into standard dumb-bell tensile bars and rectangular bars.

S. Wacharawichanant et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 21-26


23

Sample Characterization. Tensile tests were conducted according to ASTM D 638 with a universal tensile testing machine (LR 50k from Lloyd instruments). The tensile tests were performed at a crosshead speed of 50.8 mm/min. Charpy impact strength tests were performed according to ASTM D 256 at room temperature with Pendulum impact tester (Zwick/material August-Nagelstr.11.D-89079 Ulm). Each value obtained represented the average of five samples.

SEM was taken to study the morphology of the impact fracture surfaces of the SAN/-PMMA/ZnO composites and to evaluate the dispersion quality of the ZnO particles. All specimens were coated with gold before SEM study.


The tensile strength, Young’s modulus and stress at break for the composites of SAN/PMMA/ZnO as a function of composite composition are represented in Figures 1-3. The trend in variation of the tensile strength of composites at various ZnO contents is presented in Figure 1. The values of tensile strength decreased slightly with increasing ZnO content and, hence, ZnO does not improve the tensile strength of SAN/PMMA specimens prepared by compression molding.

Figure 1. Tensile strength of SAN/PMMA (20/80 wt%) blends and SAN/PMMA/ZnO composites at various contents of ZnO.

Figure 2 shows the Young’s modulus of SAN/PMMA/ZnO composites. Increasing

content of ZnO up to 1.0 wt% increased the Young’s modulus of SAN/PMMA blends. A significant increase in the Young’s modulus has been observed in composites containing ZnO due to the polar nature of the filler and the polymer promoting good interaction and dispersion between them. Addition of ZnO beyond 1.0 wt% decreased the Young’s modulus. This may be due to the aggregates of ZnO particles in a polymer matrix increased with increasing ZnO content.

Figure 2. Young’s modulus of SAN/PMMA (20/80 wt%) blends and SAN/PMMA/ZnO composites at various contents of ZnO.

The stress at break for the composites of SAN/PMMA/ZnO as a function of composite composition is represented in Figure 3. It is observed that in SAN/PMMA/ZnO compos-ites the stress at break decreased slightly with increasing ZnO content.

Figure 3. Stress at break of SAN/PMMA (20/80 wt%) blends and SAN/PMMA/ZnO composites at various contents of ZnO.



24

The Charpy impact strength for the compos-ites of SAN/PMMA/ZnO is shown in Figure 4. It is seen that the impact strength increased up to a ZnO content of 1.0 wt% for SAN/PMMA/ZnO composites, the improve-ment being due to increased energy absorpt-ion during the impact process [17]. Addition of ZnO beyond this level drastically decreases the impact strength.

The morphology of fracture surfaces of impact specimens of SAN/PMMA/ZnO composites was examined by SEM. Figures 5(a-d) show the micrographs of the impact fracture surfaces of the composites filled with 0.5, 1.0, 2.0 and 4.0 wt% of ZnO, respect-ively. It was observed that the dispersion of ZnO particles was relatively good and uniformly dispersed throughout the entire polymer matrix. However, at high ZnO

contents, the ZnO particles may aggregate to each other to become a cluster of ZnO. The dispersion of ZnO particles could have an influence on the mechanical properties of SAN/PMMA/ZnO composites.

Figure 4. Impact strength of SAN/PMMA (20/80 wt%) blends and SAN/PMMA/ZnO composites at various contents of ZnO.

(a) (b)

(c) (d)

Figure 5. SEM micrographs of SAN/PMMA (20/80 wt%) blends (a) after adding 0.5 wt% of ZnO, (b) after adding 1.0 wt% of ZnO, (c) after adding 2.0 wt% of ZnO, (d) after adding 4.0 wt% of ZnO.

S. Wacharawichanant et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 21-26


25

5. Conclusions

Composites of blends of SAN and PMMA (20/80 wt%) with ZnO were prepared by melt mixing in a twin screw extruder. The SAN/PMMA/ZnO composites showed a decreased slightly in tensile strength and stress at break with increasing filler content. Increasing content of ZnO up to 1.0 wt% increasing the Young’s modulus and impact strength of SAN/PMMA blends while the addition of ZnO beyond 1.0 wt% decreased the Young’s modulus and impact strength. The dispersion of ZnO particles in a polymer matrix was investigated using SEM. It was

observed that the dispersion of ZnO particles was relatively good and ZnO particles may aggregate to each other to become a cluster of ZnO at high ZnO contents.

Acknowledgements

The authors would like to acknowledge the financial support from the Research, Devel-opment and Engineering (RD&E) Fund through National Metal and Materials Tech-nology Center (MTEC), National Science and Technology Development Agency (NSTDA), Thailand and Silpakorn University Research and Development Institute (SURDI).

References

[1] Chae, D.W., & Kim, B.C. (2005). Character-ization on polystyrene/zinc oxide nanocom-posites prepared from solution mixing. Polymers for Advanced Technologies, 15, 846-50.

[2] Dang, Z., Fan, L., Zhao, S., & Nan, C. (2003). Dielectric properties and morphologies of composites filled with whisker and nanosized zinc oxide. Materials Research Bulletin, 38, 499-507.

[3] Xu, Y., Brittain, W. J., Xue, C., & Eby, R.K. (2004). Effect of clay type on morphology and thermal stability of PMMA–clay nano-composites prepared by heterocoagulation method. Polymer, 45, 3735-46.

[4] Yang, K., Yang, Q., Li, G., Sun, Y., & Feng, D. (2006). Morphology and mechanical pro-perties of polypropylene/calcium carbonate nanocomposites. Materials Letters, 60, 805-9.

[5] Huang, C.K., Chen, S.W., & Wei, W. C.J. (2006). Processing and property improvement of polymeric composites with added ZnO nanoparticles through microinjection mold-ing. Journal of Applied Polymer Science, 102, 6009-16.

[6] Tjong, S.C., & Liang, G. (2007). Electrical behavior of high density polyethylene/ZnO nanocomposites. e-Poly-mers, 037, 1-10.

[7] Meneghetti, P., & Qutubuddin, S. (2006). Synthesis, thermal properties and applicat-ions of polymer-clay nanocomposites. Ther-ochimica Acta, 442, 74-7.

[8] Demir, M.M., Memesa, M., Castignolles, P., & Wegner, G. (2006). PMMA/zinc oxide nanocomposites prepared by insitu bulk polymerization. Macromolecular Rapid Com-munications, 27, 763-70.

[9] Jang, B.N., & Wilkie, C.A. (2005). The effects of clay on the thermal degradation behavior of poly(styrene-co-acrylonitirile). Polymer, 46, 9702-13.

[10] Zheng, J., Ozisik, R., & Siegel, R.W. (2005). Disruption of self-assembly and altered mechanical behavior in polyurethane/zinc oxide nanocomposites. Polymer, 46, 10873-82.

[11] Lee, M.H., Dan, C.H., Kim, J.H., Cha, J., Kim, S., Hwang, Y., & Lee, C.H. (2006). Effect of clay on the morphology and proper-ties of PMMA/poly-(styrene-co-acrylonitrile) /clay nanocomposites prepared by melt mixing. Polymer, 47, 4359-69.

[12] Du, M., Gong, J., & Zheng, Q. (2004). Dynamic rheological behavior and mor-phology near phase-separated region for a LCST-type of binary polymer blends. Polymer, 45, 6725-30.

[13] Wacharawichanant, S., Thongyai, S., Tano-dekaew, S., Higgins, J.S., & Clarke, N. (2004). Spinodal decomposition as a probe to measure the effects on molecular motion in poly(styrene-co-acrylonitrile) and poly (met-hyl methacrylate) blends after mixing with a low molar mass liquid crystal or commercial lubricant. Polymer, 45, 2201-9.



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[14] Ma, X., Tang, L., Zhou, B., Xiao, Y., Lei, H., & Wang, Z. (2010). Preparation of ZnO/PMMA inorganic/organic com-posite microspheres via soap less emul-sion polymerization. e-Polymers, 046, 1-5.

[15] Jin, J., Wu, J., & Frischknecht, A.L. (2009). Modeling microscopic morpho-logy and mechanical properties of block copolymer/nanoparticle composites. M-acromolecules, 42, 7537-44.

[16] Huang, H.-X., & Zhang, J.-J. (2009). Effects of filler-filler and polymer–filler interactions on rheological and mech-anical properties of

HDPE–wood composites. Journal of Applied Polymer Science, 111, 2806-12.

[17] Wacharawichanant, S., Thongyai, S., Phutthaphan, A., & Eiamsam-ang, C. (2008). Effect of particle sizes of zinc oxide on mechanical, thermal and mo-rphological properties of polyoxymet-hylene/zinc oxide nanocomposites. Po-lymer Testing, 27, 971-6.




Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 27-34

Research Article

Study of Carbonyls-TiO2 as Co-Catalysts in Photo-Oxidative Degradation of Hydrocarbon Backbone

T. Manangan1,2*, S. Shawaphun1,2, K. Kasetkulasheep1

1 Department of Industrial Chemistry, Faculty of Applied Science, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand.

2 Research Center of Nano Industries and Bio-plastics, King Mongkut’s University of Technology North Bangkok, Bangkok 10800, Thailand.


1. Introduction

Due to their useful mechanical properties and physical properties applicable for various utilizations, polyethylene (PE) and polypro-

pylene (PP) are the most popular plastics in packaging industries. However, these plastics take up to 450 years to decompose [1]. Therefore, enormous amount of these plastic wastes has been disposed into the environ-ment and caused serious natural resource contamination every year. The most serious plastic waste problem was plastic carrier bags which were used about 8,000,000 bags

Abstract

In present, environmental problems have been dramatically increasing especially caused by packaging plastics such as polyethylene and polypropylene. Due to their versatile functionalization, acetophenone and benzophenone derivatives using as plastic additives were synthesized to preliminarily study the rate of plastic photo-oxidative degradation under ultraviolet irradiation with various sizes (micro-sized and nano-sized) and crystal structures (rutile and anatase) of titanium dioxide (TiO2) co-catalyst by monitoring the carbonyl index. In benzene solution, the acetophenone derivatives showed significant photolytic behavior especially under the shortwave 254-nm ultraviolet irradiation while the benzophenone derivatives did not. However, in hexane solution, the benzophenone derivatives were able to induce the oxygenation of hexane and increase carbonyl index as well as the acetophenone derivatives. Using these carbonyl derivatives as co-catalysts with titanium dioxides most particle sizes and crystal structures provided excellent photo-oxidative degradation of the hydrocarbon backbone within 96 hours by several folds and up to 100 times with benzophenone derivatives. However, titanium dioxide particle size did not show much advantage possibly due to the mobilization of the free radical intermediates in the solution. In conclusion, this co-catalyst system could be used to introduce photo-degradation of the conventional hydrocarbon based plastic packaging.

Keywords: Photo-catalysts, Degradation, Oxo-degradable, Titanium dioxide.



28

annually in a typical developed country. Up to 80% of them end up in nature. Various regulations, policies and managements have been used intensively in many countries worldwide to overcome these problems. Recently, bio-plastics and biodegradable plastics have also been brought up to solve such problems [2]. However, the large scale production of bio-plastics has not yet been economical and easily accessible. The rapid and affordable way is to modify the convent-ional plastics and make them degradable after used. In order to do this, the plastics additives were blended into the plastics as an oxidative degradation catalysts triggered mostly by light, heat or chemicals. In our previous work, the oxo-degradability has been intro-duced in situ using metal oxide photo-catalytic additives that can also act as the thermal oxidative catalyst generating carbonyl com-pounds during the thermal plastic film processing. Among many other metal oxides, titanium dioxide (TiO2) was found to give excellent oxidative catalytic activity both thermally and photolytically [3]. In addition, due to its superb characteristics such as affordable, non-toxicity and stability, the TiO2 has become the excellent choice for photo-catalyst in order to make the convent-ional packaging plastics such as PE and PP become degradable after used, especially for food packaging plastics [4]. Furthermore, the carbonyl compounds alone can also be used as the oxo-degradable photo-catalyst of PE and PP degradation [5-7]. In this paper, the co-catalytic behaviors of various TiO2 and various carbonyls have been investigated.

2. Theory

Typically, the current available options for PE and PP waste treatments were the tradit-ional landfill, the high-energy consumed incineration, 3R protocol (Reduce, Reuse and Recycle) and composing. All of the above, however, can be done only if the plastics can be collected and separated. Plus, the waste management and energy consumption can be costly. The auto-oxidative degradation of PE and PP initiated by light (so called photo-oxidative degradation) has been brought up in

order to deal with the plastic waste after used with out any hidden cost or management. The photo-oxidative plastic degradation consists of two major steps, photo-oxidation and photolysis. The first step is usually the rate determining step because the plastics have to react with oxygen in the air and form the unstable peroxide adducts which can then undergo photolysis to give carbonyl and other free radical intermediates e.g. alkyl radicals or hydroxyl radicals [8]. Moreover, the carbonyl intermediates have been known to be very good chromophores and can be photolyzed to more free radicals [9]. The radicals then undergo propagation step in the chain reaction of plastic degradation and give more of oxygenated carbon backbones in the plastics in forms of hydroxyls, aldehydes, ketones and carboxylic acids. These oxo-degradable intermediates then can be decom-posed biologically at low molecular weight ultimately resulting carbon dioxide, water and biomass [10-12].

The objective of this research is to study the possibility to accelerate the PE and PP degradation under solar light exposure after used and casted away using TiO2 and carbonyl co-catalyst system. The nano-sized TiO2 particle was also used in this investi-gation in comparison with the commercial and the micro-sized. The acetophenone, benzophenone and their derivatives were synthesized and used as co-catalysts. The photo-catalytic oxidative activity of the co-catalyst systems was done in both benzene and hexane using ultraviolet light irradiation at average wavelength at 254nm and 366nm. The oxidation then was monitored via the carbonyl index measurement thoroughly to understand their catalytic degradation path-ways.


TiO2 (analytical grade) referred as commercial TiO2, nano-sized TiO2 (rutile, particle size less than 100 nm, SSA more than 14 m2/g), nano-sized TiO2 (anatase, particle size less than 25 nm, SSA in range of 200-220 m2/g), acetophenone (analytical grade),



29

benzophenone (analytical grade), benzene (HPLC grade) and hexane (HPLC grade) were purchased from Sigma Aldrich®. The micro-sized TiO2 (particle size less than 63 µm) was prepared by particle size sieving at 230 Mesh. Most catalysts were oven dried for 24 hours and kept in desiccators before use.

Preparation of Carbonyl Co-Catalysts. 3-Bromoacetophenone, 3-bromobenzophenone, 3-nitroacetophenone and 3-nitrobenzophen-one were synthesized and fully characterized as described in previous communication [5].

Photolytic Behaviours of Carbonyls. The dry 10-ml solutions of acetophenone, benzophe-none, 3-bromoacetophenone, 3-bromobenzo-phenone, 3-nitroacetophenone and 3-nitro-benzophenone were prepared in benzene at 1%, 2%, 3%, and 5% (w/v) concentrations in 2mm-thick pyrex sealed tubes. After vigor-ously well-shaken, the solutions were irradiated under a 20-watt shortwave UV lamp (average wavelength at 254 nm) and a 20-watt long wave UV lamp (average wavelength at 366 nm) at 30 cm in distance. The solution carbonyl index (CI) was monitored daily by taking out 1-ml aliquot at 24, 48, 72 and 96 hour for FTIR spectrum using a sodium chloride cell [9].

Photo-Oxidative Activity of Carbonyls. The experimental procedure was done exactly the same as above except all carbonyl solutions were prepared in hexane.

Catalytic Photo-Oxidative Activity of Various TiO2 Catalysts. The powders of commercial micro-size TiO2 (predominantly rutile), nano-sized TiO2 (anatase) and nano-sized TiO2 (rutile) were individually mixed into both benzene and hexane at 1%, 2%, 3%, and 5% (w/v) concentrations. After vigorously well-shaken, the mixture FTIR spectra were scanned and the mixture carbonyl index was determined as described above.

Photo-Oxidative Behaviours of Various TiO2 and Carbonyl Co-Catalytic Systems. The vigorously shaken anhydrous hexane solut-ions of various combinations of the TiO2 catalysts at 1% and 2% (w/v) and the carbonyl co-catalysts at 1%, 2%, 3% and 5% (w/v) in 2mm-thick pyrex sealed tubes were irradiated under a 20-watt UV lamps as previously described. The solution carbonyl index (CI) was then monitored daily with the same method above.


Photolytic Activity of Carbonyls. The carbo-nyl index profiles of all carbonyls used in this study can be categorized into 2 groups: i) Norrish type I photoreaction in which the carbonyl interacts with light and undergoes homolysis to give radicals and ii) Norrish type II photoreaction in which the carbonyl can only be excited by light and introduce other reactions without molecular photolysis. The UV irradiation result in benzene solution showed that acetophenone and 3-bromo-acetophenone showed significant photolytic behavior in the first 24-hour, especially under the shortwave UV254-nm irradiation as shown in Figure 1. Then the carbonyl index tended to rise up to the same value or slightly higher. Under the longwave UV-366 nm irradiation, this behavior was barely notice-able as shown in Figure 2. The nitroaceto-phenone also showed similar photolytic

Figure 1. Photolytic profile of benzene solutions of acetophenone 1–5% (w/v) under UV254-nm irradiation.



30

Figure 2. Photolytic profile of benzene solutions of acetophenone 1–5 % (w/v) under UV366-nm irradiation.

profile with less change, even though it processes high molar absorptivity (results not shown here). From these results, it is possible to conclude that acetophenone and its deriv-atives in this study interact with light through Norrish type I photoreaction.

The results of benzophenone derivatives in benzene solution in the other hand did not show any change in carbonyl index under both shortwave and longwave UV irradiation as shown in Figure 3. This indicated that benzophenone and its derivatives interact with light through Norrish type II reaction. However, this type of molecule can also act as the sensitizer which only absorbs the light

at ground state and becomes excited. The energy stored in the excited state then can be transferred to another molecule and generate further photoreactions. This mechanism was used widely in many photo-catalytic applicat-ions.

Photo-Oxidative Activity of Carbonyls. Hexane solution was used as a hydrocarbon backbone in order to investigate the photo-lytic activity of the carbonyls derived from acetophenone (Norrish type I) and benzo-phenone (Norrish type II). The experimental procedure was done exactly the same as above except all carbonyl solutions were prepared in hexane. Again, most photo catalytic reactions under the shortwave UV irradiation showed about 2-3 folds higher reactivity than photoreaction under the longwave irradiation. Also, the rate of photo-oxidation of hexane increased sharply in the fourth day as shown in Figure 4. Moreover, the higher catalysts concentration gave the more increment of carbonyl index in hexane regardless of the photo-catalytic mechanism. This indicated that the carbonyl catalysts enhance the rate of oxygenation of hexane to give more oxo-degradable products. From this result, photo-oxidative degradation of PE and PP films using acetophenone and benzophenone derivatives provided great catalytic activity as reported in our previous communication [5].

Figure 3. Photolytic profile of benzene solutions of benzophenone 1 – 5% (w/v) under UV254-nm irradiation.



31

Figure 4. Photo-oxidation of hexane using acetophenone and benzophenone as catalysts under UV254-nm irradiation.

Figure 5. Photo-oxidation of hexane using 3-bromoacetophenone and 3-bromobenzophenone as catalysts under UV254-nm irradiation.

Surprisingly, 3-bromoacetophone and 3-bromobenzo phenone showed similar results to the nitro derivatives as shown in Figure 5. These carbonyls did not show any rapid photo-catalytic activity in the first 4 days. However, the carbonyl index continued to increase steadily.

Catalytic Photo-Oxidative Activity of Various TiO2 Catalysts. The carbonyl index of the benzene solutions containing the powders of commercial micro-size TiO2, nano-sized TiO2

(anatase) and nano-sized TiO2 (rutile) under UV irradiation did not provide any catalytic behaviour. In hexane solutions, however, suggested catalytic photo-oxidation of the hexane carbon backbone. The particle size effect became so clear when the nano-sized TiO2 was used. The nano-sized TiO2-anatase provided great photo-oxidative activity by 2-2.5 folds over the commercial TiO2. This is probably due to dispersion effect and specific surface area.



32

Photo-Oxidative Behaviours of Various TiO2 and Carbonyl Co-Catalytic Systems. When above TiO2 photo-catalysts were used in present of carbonyls installed with chromo-phore, most photo-oxidation of hexane was catalytically enhanced by several folds. Applying acetophenone (2% w/v) with the nano-sized TiO2-anatase 1 and 2%w/v, the carbonyl index increased up to 4.1 and 5.3, respectively as shown in Figure 6. Similarly, when benzophenone was used with both nano-sized TiO2, the carbonyl index dramati-cally raised to 9.4–21.1 in 96 hours as shown

in Figure 7.

Surprisingly, 3-bromoacetophenone and 3-bromobenzophenone showed strangely high catalytic oxidation activity through all concentration as shown in Figure 8 and 9. This could be some types of side reactions or unexplainable mechanisms generated via C-Br bond cleavage. However, it is important to note that the bromo compounds might provide photo-oxidation and photolysis of hydrocarbons.

Figure 6. Photo-oxidation of hexane using acetophenone (2%) as co-catalyst with various TiO2 under UV254-nm irradiation.

Figure 7. Photo-oxidation of hexane using benzophenone (3%) as co-catalyst with various TiO2 under UV254-nm irradiation.



33

Figure 8. Photo-oxidation of hexane using 3-bromoacetophenone (5%) as co-catalyst with various TiO2 under UV254-nm irradiation.

Figure 9. Photo-oxidation of hexane using 3-bromobenzophenone (3%) as co-catalyst with various TiO2 under UV254-nm irradiation.

5. Conclusions

The photolytic activity of the acetophenone derivatives was investigated in benzene solution in order to understand the catalytic behavior. The photolytic behavior of aceto-phenone derivatives only was observed, especially under the shortwave 254-nm ultraviolet irradiation. The photoreaction of all acetophenone derivatives was found to occur via Norrish type I reaction which the carbonyl index was initially reduced and then

gained back up to a plateau at the original value. The benzophenone derivatives in the other hand underwent possibly via Norrish type II photoreaction or sensitization in which they did not show significant change of carbonyl index in benzene. However, in hexane solution, the benzophenone deriva-tives were able to induce the oxygenation and increase carbonyl index as well as the acetophenone derivatives. Photo-oxidation of hexane using only various TiO2 suggested that good activity could be achieved only



34

through the nano-sized TiO2 anatase. Using the carbonyl derivatives as co-catalysts with these TiO2 in all particle sizes and crystal structures provided excellent catalytically photo-oxidative degradation of the hydrocar-bon backbone within 96 hours by several folds and up to 100 times with benzophenone derivatives. However, titanium dioxide parti-cle size did not show much advantage possi-bly due to the mobilization of the free radical intermediates in the solution. In conclusion,

this co-catalyst system could be used to introduce photo-degradation of the convent-ional hydrocarbon based plastic packaging such as PE, PP and PS.

Acknowledgements

The authors would like to thank the National Research Council of Thailand (NRCT) for the financial support of this project.

References

[1] Guillet, J., In: Gilead D. Scott, G., editors. Degradable Polymers Principles & Applicat-ions. London: Chapman & Hall; 1995.

[2] Sudesh, K., Abe, H. & Doi, Y. (2000). Synthesis, structure and properties of polyhy-droxyalkanoates: biological polyesters. Pro-gress in Polymer Science, 25, 1503-55.

[3] Shawaphun, S., Manangan, T., & Wach-aravichanant, S. (2010). Thermo-and photo-degradation of LDPE and PP films using metal oxides as catalysts, Advanc-ed Materi-als Research, 93-94, 505-9.

[4] Zhao, X., Li, Z., Chen, Y., Shi, L., & Zhu, Y. (2007). Solid-phase photocatalytic degradat-ion of polyethylene plastic under UV and solar light irradiation. Journal of Molecular Catalysis A: Chemical, 268, 101-6.

[5] Manangan, T., Shawaphun, S., & Wach-aravichanant, S. (2010). Acetophenone and benzophenone derivatives as catalysts in photo-degradation of PE and PP films. Advanced Materials Research, 93-94, 284-7.

[6] Roy, P.K., Surekha, P., Rajagopal, C., Cahtterjee, S.N., & Choudhary, V. (2007). Studies on photo-oxidative degra-dation of LDPE films in the presence of oxidased polyethylene. Polymer Degradation and Stability, 92, 1151-60.

[7] Chatgilialoglu, C., Ferreri, C., & Sommazzi, A. (1996). Free Radical Carbonylation of 1, 4-cis-Polybutadiene. Journal of American Chemical Society, 1996; 118: 7223-4.

[8] Gijsman, P., Meijers, G., & Vitarelli, G. (1999). Comparison of the UV-degradation chemistry of polypropylene, polyethylene, polyamide 6 and polybutylene terephthalate. Polymer Degradation and Stability, 65, 433-41.

[9] Chiellini, E., Corti, A., D’Antone, S., & Baciu, R. (2006). Oxo-biodegradable carbon backbone polymers- Oxidative degradation of polyethylene under accelerated test condit-ions. Polymer Degradation and Stability, 91, 2739-47.

[10] Haines, J.R., & Alexander, M. (1975). Microbial degradation of high-molecular wei-ght alkanes. Applied Microbiology, 28, 1084-5.

[11] Jakubowicz, I., Yarahmadi N., & Peterson, H. (2006). Evaluation of the rate of abiotic degradation of biodegradable polyethylene in various environments. Polymer Degradation and Stability, 91, 1556-62.

[12] Jakubowicz, I. (2003) Evaluation of degrada-bility of biodegradable poly-ethylene (PE). Polymer Degradation and Stability, 80, 39-43.


Sci. J. UBU, Vol. 1, No. 2 (July – December, 2010) 35-39



Research Article

Characteristics of TiO2-SiO2 Microparticle Composites Using Different Types of SiO2 Particle

J. Janlamool , B. Jongsomjit*

Center of Excellence on Catalysis and Catalytic Reaction Engineering, Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok

10330, Thailand.


1. Introduction

The study of hybrid particles can provide important fundamental insights for new func-tional materials, such as photonic catalyst [1], high-performance electronic materials [2] and so on. Nowadays, the titania-silica compos-ites supported metal catalysts have attracted the scientist attention in the catalysis field. Recently, it was reported that titania-silica composites particles exhibited better catalytic properties than classical oxides, such as

titania and silica [3]. The inclusion of titania in silica structures is reported in several works. Eidenmeyer et al. [4] grafted metal organic titanium reagents onto the dehydrated MCM48 samples in dried hexane. Xiao et al. [5] produced mesoporous titanosilicates with high catalytic activity. Ogawa [6] produced nanoporous silica films containing titanium. Mrowiec-Bialon [7] synthesized titania–silica aerogels. Previous research prepared TiO2-SiO2 composite microspheres with micropor-ous SiO2 core and mesoporous TiO2 shell structures by hydrolysis of titanium tetrabutyl ortho-titanate in the presence of microporous silica microspheres using hydroxypropyl-cellulose as a surface esterification agent and

Abstract

The TiO2-SiO2 microparticle composites were prepared by deposition of TiO2 particles on the spherical silica particle (SSP) and MCM41 surface using hydrolysis of titanium isopropoxide to obtain TiSSP and TiMCM, respectively. The TiO2-modified metal-surface interaction of SiO2 exhibited the sufficient surface area and pore volume, even decrease with increased calcination temperature. The anatase crystallites of titania on TiSSP and TiMCM began to form at 650 C. At calcination temperature of 900 C, the appearance of a portion of TiO2 had the form of rutile, which was only found in the TiSSP. However, the surface areas of TiSSP and TiMCM calcined at 900 C were remarkably low at 11 and 56 m2/g, respectively. The XRD patterns and DTA/TG indicated that TiMCM had better thermal stability than TiSSP. The titania distribution of both samples was uniform.

Keywords: Titania, Silica, MCM41, Hydrolysis, Phase transformation.



36

porous template, and then dried and calcined at different temperatures [8].

The TiO2-SiO2 composites supports can exhi-bit the novel properties that are not finding in a single oxide supports, with combination, the benefit of TiO2 support provided the thermal stability and SiO2 has a sufficiently high surface area. The aim of this research focuses on synthesis of the titania-silica composites using hydrolysis of titanium isopropoxide with spherical silica particle and MCM41. The samples were characterized by several techniques, such as differential ther-mal analysis and thermogravimetric (DTA-/TG), X-ray diffraction (XRD), Brunaner-Emmelt-Teller (BET) surface area using the nitrogen physisorption. The morphology and TiO2 distribution of composite particles were determined by scanning electron microscopy (SEM) and energy dispersive X-ray spectro-scopy (EDX).


Materials. Titanium isopropoxide 97% (TiPOT) and tetraethyl othosilicate 98% (TEOS) were obtained from Aldrich. Ammo-nia 30% was available from Panreac. Ethanol 99.99% was available from J.T. Baker. Isopropanol was supplied from QReC.

Preparation of the Spherical Silica Particle (SSP) and MCM41. The composition of the synthesis gel had following molar ratio: 1 TEOS : 0.3 C16TMABr : 11NH3 : x Ethanol : 144 H2O. Molar ratios of ethanol addition were 0 and 58 for the preparation of MCM41 and SSP, respectively. The solution was further stirred for 2 h at room temperature. The white precipitate was then collected by filtration and washed with distilled water. Dried samples were calcined at 550 C for 6 h with a heating rate of 10 C min−1 in air. Preparation of the TiO2-SiO2 Composites Particles. 0.917 g of TiPOT (ca. 25 wt% of TiO2) was diluted in 3 g of isopropanol. Then, 0.75 g of silica from 2.2 was added to the solution and stirred for 1 h. Hydrolysis

was performed by addition of ammonia (H2O : TiPOT = 4:1). The sol was further stirred for 20 h at room temperature. Then, the sample was dried at 110 C for 24 h. Finally, the samples were calcined at 550, 650, 750 and 900 C for 2 h in a muffle furnace.

Characterization. The morphology of the samples was characterized by SEM/EDX (mode JSM-5800LV). DTA/TG was perfor-med with Shimadzu TGA model 50 at heating rate of 10 C/min from room temper-ature to 1000 C. The XRD patterns were obtained on a SIEMENS D 5000 X-ray diffr-actometer. Brunaner-Emmelt-Teller (BET) surface area and pore parameter were determined with nitrogen physisorption (N2 adsorption at 196 C in a Micro-meritics ASPS 2020).


XRD. Figure 1(a) shows the XRD patterns of TiO2/SSP (TiSSP) microparticle composites calcined at different tempera tures. At 550 C, the XRD patterns exhibited only amor-phous silica and titania resulting in broad peak observation. At 650 and 750 C, the slight diffraction peak of TiO2 crystalline can be observed at 25.3. This demonstrates that TiO2 had a small crystalline size of anatase form. The XRD patterns for two TiO2 crystalline forms (anatase, 2θ of 25.3, 48, 54, 55 and 75 ; rutile, 2θ of 27.5, 36, 41, 54.5 and 56.5) displayed distinctly at 900 C. A portion of TiO2 had the form of rutile. The ratio of rutile:anatase is 3:97 based on Scherrer equation described elsewhere [9].

The XRD patterns of TiO2/MCM41 (TiM-CM) microparticle composites is shown in Figure 1(b). At 550, 650 and 750 C, the XRD patterns of TiMCM were similar with those of TiSSP. In contrast, the XRD patterns for TiMCM exhibited only crystalline anatase phase of TiO2 at 900 C. This can be attributed to more thermal stability of TiMCM (no rutile was formed). At low calcination temperature, the crystallite size of

J. Janlamool et al., Sci. J. UBU, Vol. 1, No. 2 (July – December, 2010) 35-39


37

Figure 1. XRD patterns of TiO2-SiO2 microparticle composites calcined at 550, 650, 750 and 900 C for 2 h ; a) TiSSP and b) TiMCM.

Ti is less than 5 nm (not detectable by XRD). However, the size is more than 5 nm upon high calcination temperature (900 oC) due to agglomeration of crystal.

SEM/EDX. Figure 2 shows the SEM micro-graphs and EDX of the titania/silica micro-particle composites. Figure 2 (a) reveals the titania colloid deposition on the surface of spherical silica core in TiSSP. As a result, the slight portion of TiSSP microparticle was coagulated. The EDX mapping indicates that the titania distribution on the spherical silica surface is uniform. Figure 2 (b) shows the titania colloid deposition on the surface of MCM41 in TiMCM microparticle that was also substantial coagulation. However, the EDX mapping indicates that the titania distribution on the MCM41 silica surface is also uniform as seen for TiSSP.

DTA/TG. Figure 3 indicates that the DTA/TG curve of titania/silica microparticle composites is distinct with different types of silica. Figure 3 (a) shows the DTA curve of TiSSP that displays several endothermic peaks below 450 C. This is due to the evaporation of physical adsorbed water and isopropanol solvent. Two noticeable exothe-rmal peaks at 612 and 948 C were related to the XRD patterns as mentioned above. At 612 C, the XRD patterns indicated the exothermic peak that was due to the phase transformation of amorphous to anatase. Furthermore, the exothermal peak is caused by the phase transition from anatase to rutile at 948 C. The TG curve shows a largest weight loss about 15.84% in TiSSP. The DTA curve of TiMCM is shown in Figure 2(b). DTA curve of TiMCM displays several endothermic peaks below 450 C. This result is similar to those of TiSSP. In contrast with TiSSP, TiMCM has only exothermal peak at 645 C. The TG curve displays a weight loss about 14.93% in TiMCM.

Surface Area and Pore Parameter. Table 1 shows the BET surface area and pore volume of TiO2-SiO2 micro particle compos-ites decreases with increased calcination temperatures. At 750 C, the BET surface area and pore volume apparently decreased. This is probably due to increase of amount of TiO2 in the sample and particle agglomer-ation. However, at 900 C, the BET surface area and pore volume of TiSSP and TiMCM were remarkably low at 11 and 56 m2/g, respectively. This can be attributed to grain growth from phase transformation of anatase to rutile and the particles sintering. It is reported that the micro-structures of titania particle calcined at various temperatures can be determined by high resolution scanning electron microscopy. The smaller anatase crystallites grow into bigger rutile crystal-lites through the phase transformation, lead-ing to the decrease of the voids among anatase crystallites [10]. As the result the surface area and pore volume remarkably decrease after calcination at 900 C.

(a)

(b) A

A

R

A A A

R A A A

A : anatase R : rutile



38

Figure 2. SEM micrographs of a) TiSSP and b) TiMCM microparticle compostes calcined at 750 C.

Figure 3. DTA/TA curves of TiO2-SiO2 microparticle composites ; a) TiSSP and b) TiMCM.

Table 1. BET surface area, pore volume, and pore diameter of support samples.

Samples ABET (m2/g) Vp (cm3/g) DBJH (nm) Pure SSP 927 0.8135 2.04 TiO2/SSP calcine 750 632 0.3425 2.71 TiO2/SSP calcine 900 11 0.0365 11.68 Pure MCM41 1187 1.0287 2.13 TiO2/MCM calcine 750 323 0.1764 3.17 TiO2/MCM calcine 900 98 0.0593 5.43

948

612

(b)

645

(a)

(a) Ti

Ti(b)

J. Janlamool et al., Sci. J. UBU, Vol. 1, No. 2 (July – December, 2010) 35-39


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5. Conclusions

The TiO2-SiO2 microparticle composites were prepared by deposition of TiO2 particles on the spherical silica particle and MCM41 surface using hydrolysis of titanium isopro-poxide. The TiO2-SiO2 microparticle com-posites provided the higher surface area than pure titania at high temperature. The results showed that the distribution of titania on the

silica surface was uniform. The TiMCM had better thermal stability than the TiSSP as proven by the XRD and DTA/TG measure-ment.

Acknowledgements

Authors thank the Thailand Research Fund (TRF) for the financial support of this project.

References

[1] Fu, W., Yang, H., Li, M., Yang, N., & Zou, G. (2005). Anatase TiO2 nanolayer coating on cobalt ferrite nanoparticles formagnetic photo catalyst. Mater. Lett. 59, 3530–4.

[2] Chung, H.T., Cheong, D.S., & Kim, C.S. (2005). Role of nanoparticles in PNN-PZT/Ag nanocomposite. Mater. Lett. 95, 920–4.

[3] Fu, X.A., & Qutubuddin, S. (2001). Prepara-tion and Characterization of Titania Nano-coating on Mono disperse Silica Particles Colloids Surf. A 178, 151.

[4] Eidenmeyer, M., Grasser, S., Köhler, K., & Anwander, R. (2001). Microporous Mesopor-ous matter. 3276, 44-5.

[5] Xiao, F.S., Han, Y., Yu, X., Meng, M., Yang, S., & Wu, J. (2002). Hydro thermally stable ordered mesoporous titanosilicates with highly active catalytic sites. J. Am. Chem. Soc. (6), 888.

[6] Ogawa, M. (2003). Nanoporous silica films containing aluminum and titanium. Colloid and Polymer Science, 281 (7), 665-72.

[7] Mrowiec Białoń, J. (2000). Porosity of titania-silica aerogels prepared with ammonium fluoride. Polish Journal of Chemistry, 74 (4), 539-48.

[8] Zhao, L., Yu, J., & Cheng, B. (2005). Preparation and characterization of SiO2/TiO2 composite microsphere with microporous SiO2 core/meso porous TiO2 shell. Solid state Chemis-try, 178, 1818-24.

[9] Jongsomjit, B., Wongsalee, T., & Praserthdam, P. (2005). Characteristics and catalytic pro-perties of Co/TiO2 for various rutile:anatase ratios. Catalysis Communication, 6, 705-10.

[10] Kumar, K.N.P. (1994). Porous nanocompos-ites as catalyst supports: Part I. ‘second phase stabilization’, thermal stability and anatase-to-rutile transformation in titania-alumina nano composites. Applied Catalysis A. General, 119, 163-83.


Sci. J. UBU, Vol. 1, No. 2 (July – December, 2010) 40-45 SCIENCE JOURNAL Ubon Ratchathani University http://scjubu.sci.ubu.ac.th


Research Article

Synthesis of Carbon Microspheres from Starch by Hydrothermal Process

S. Ratchahat1, N. Viriya-empikul2, K. Faungnawakij2, T. Charinpanitkul1, A. Soottitantawat1,*

1Center of Excellence in Particle Technology, Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok 10330, Thailand.

2National Science and Technology Development Agency, 130 Thailand Science Park, Paholyothin Rd., Klong Luang, Pathumthani 12120, Thailand.


Abstract

This study showed a facile catalyst-free method to synthesize carbon microspheres (CMSs) via hydrothermal and carbonization process using various types of starch as starting materials. In hydrothermal process, starch was hydrolyzed, dehydrated, and polymerized to form carbon microspheres in water as a medium without involving any hazardous solvents. After hydrothermal process, the dried products were treated by heat in carbonization process under nitrogen atmosphere to develop their pore system of carbon microspheres. The two main types of starch, modified starch and native starch, were employed to address differences in particle size and morphology of resulting carbon microspheres. The SEM images clearly illustrated that the carbon microspheres have their perfect spherical morphology and smooth surface. The particle size distributions of the products with a size range of 0.4-4.0 µm were determined by laser diffraction technique (Mastersizer). The particle size and particle size distribution of carbon microspheres strongly depended on types of starch. In other words, carbon microspheres from modified starch tended to smaller in particle size than carbon microspheres from native starch because of water-solubility of modified starch higher than native starch. After carbonization process, structural CMSs characterization performed by X-ray diffraction technique (XRD) indicated semi-hexagonal graphite structures which would be suitable for secondary lithium ion application. Elemental compositions of the carbonaceous products determined by energy dispersive X-ray spectroscopy (EDX), indicated that a main component was carbon being inert to many chemical reactions. Furthermore, these carbon materials have specific BET areas in the range of 400-500 m2/g which were formed during carbonization process. All N2 adsorption-desorption isotherms of these carbon materials have type I isotherm regarding to IUPAC classification that indicated micropore system.

Keywords: Carbon microspheres, Starch, Porous carbon, Hydrothermal process.

S. Ratchahat et al., Sci. J. UBU, Vol. 1, No. 2 (July – December, 2010) 40-45


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1. Introduction

Since the significant finding of Buckminster-fullerene (C60) [1] and carbon nanotubes (CNTs) [2] considerable efforts have been made toward the synthesis of functional carbonaceous materials with diverse morpho-logies and structures, such as colloidal spheres [3], nanofibers [4], coin-like hollow carbons [5], macroflowers [6], and so on. Among the different morphologies of carbon-iceous materials, carbon microspheres (CMSs) have attracted widespread interest, owing to their potential properties in adsorb-ents [7], catalyst supports [8], and anode material for lithium ion batteries [9] and templates for fabricating core-shell or hollow structures [10]. The CMS particles have been synthesized by many techniques, such as pressure carbonization [11], chemical vapor deposition [12], mixed-valence oxide-catal-ytic carbonization [13], and reduction of carbides with metal catalysis [5]. Accord-ingly, various applications have been intens-ively developed [14]. Carbon microspheres (CMSs) with a perfect shape have the priority in catalyst support [15] and template utilization [16]. However, there are a few reports of CMSs with a uniform size and perfect spherical morphology from various types of starch. Consequently, in this study the synthesis of CMSs from various types of starch was investigated via hydrothermal process and following by carbonization pro-cess. The aim of this study is to investigate effects of types of starch on particle size and morphology of carbon microspheres. The two main types of starch, modified starch and native starch, were used to investigate differ-ences in particle size and morphology of the obtained carbon microspheres. After hydro-thermal process, the dried CMSs had been carbonized under nitrogen atmosphere. The carbonization process had highly developed microporosity of CMSs but had removed the reactive functional group (-OH,-COOH) on their surface [17]. The porous CMSs have highly microporosity and inert surface that can be used as adsorbents or gas storage materials. Without carbonization process, the CMSs have the reactive functional group on

CMSs surface which can be immobilize target reactive agents on the surface without further surface modification. The uniform CMSs can be determine particle morpho-logies and particle size distributions by scan-ning electron microscopy (SEM) and laser diffraction method (Mastersizer 2000), resp-ectively. The porous CMS particles were characterized by X-ray diffraction method (XRD) to reveal their crystalline properties. They were determined their specific surface area both before and after carbonization process to reveal the development of porous structure using adsorption–desorption of nitrogen or the Brunauer, Emmett, Teller method (BET method). Moreover, elemental components of the porous CMS particles were determined by energy dispersive X-ray method (EDX) to demonstrate carbon content in their structure.


Materials. Two types of modified starch, Hi-CAP®100 and CAPSUL®, were obtained from National Starch and Chemical Ltd, (Bangplee, Thailand). These starches (HI-CAP®100 and CAPSUL®) were partially hydrolyzed of waxy maize starch and then derivatized to impart lipophilic properties with n-octenyl succinic anhydride. They can be immediately dissolved in water. The difference in structure of HICAP®100 and CAPSUL®, HICAP®100 is a straight chain starch but CAPSUL® is a branch chain starch. Therefore HICAP®100 has water-solubility more than CAPSUL®. Meanwhile, other native starch or crystallized starch (corn, tapioca, wheat, rice, and sticky rice starch) were obtained from a general commercial resource. High purity nitrogen gas (99.999%) was purchased from TIG. Synthesis of the CMSs. In the hydrothermal process, starch (5.0 g) was dispersed in 45.0 mL of de-mineralized water and magnetically stirred at 60C for 30 min. The mixture was filled in a 50 mL Teflon-lined stainless autoclave. Subsequently, the autoclave was put into an oven, which was heated at 180C



42

for 12 h. The autoclave was cooled to room temperature naturally. Dark precipitates were collected and washed with de-mineralized water several times and dried in an oven at 100C for 24 h. The obtained powders were carbonized in a tube furnace under N2 atmosphere. The N2 flow rate, final temper-ature and heating rate of the furnace were 100 ml/min, 600C and 1C/min, respectively. Characterization. The particle size distribut-ions of synthesized carbon microspheres were determined by laser diffraction techn-ique (Mastersizer 2000: Malvern, United Kingdom). The samples were characterized by X-ray powder diffraction (XRD, SIEMENS D5000, Japan) using CuK radiation. The morphology observation of the samples was examined with scanning electron microscopy (SEM, JEOL: JSM-5410LV, Japan). The specific BET surface area was measured by N2 adsorption- desorption at -196C (BEL: BELSORP-mini, Japan). The carbonaceous products were confirmed by energy-dispersive X-ray spectra (EDX).

3. Results and Discussion.

Figure 1 shows the typical morphology of the synthesized CMSs from hydrothermal process. Perfect spherical shape and smooth surface can be observed in CMSs from all types of starch. These spherical particle formations are generally patterns in particle formation because they can keep the lowest their surface energy in spherical formation. When HI-CAP®100 was used as a carbon precursor, the smallest size and highly uniform CMSs were obtained (Figure 1a, b) because of its high water-solubility. Therefore HI-CAP®100 can be hydrolyzed immediately to form simultaneous nucleates of carbon microspheres. Nonetheless, the CMSs obtained from CAPSUL® showed the big particle size more than carbon microspheres from HI-CAP®100 because CAPSUL® which is a branch modified starch can continuously be hydrolyzed to form a shell after nucleates forming. In other words,

shell growth formation plays an important role than nucleate formation. On the other hand, if native starches (unmodified starches) were used as starting material, the CMSs became larger and some of CMSs became aggregates with diameters ranging from 1.0 to 7.0 μm (Figure 1f-h). In this work, it appears that HI-CAP®100 could provide monodisperse CMSs when compared with other carbon sources. However, some of unmodified starches (corn and tapioca starch) can also result in the monodisperse CMSs (Figure 1d, e).

Based on our experimental results, CMS particle size and its distribution depended on types of starch (Figure 2). These particle size

Figure 1. SEM images of CMSs after carbonization: (a) HI-CAP®100; (b) HI-CAP®100 at 10,000 magnification; (c) CAPSUL®; (d) corn; (e) tapioca; (f) sticky rice; (g) wheat; and (h) rice starch.



43

2 (degree)

(002

)

(10

1)

Inte

nsit

y (a

.u.)

HI-CAP® 100

Tapioca

CAPSUL® Sticky rice

distributions directly related to SEM observ-ations. The particle size distribution of CMSs from HICAP®100 has the narrowest because HICAP®100 has its high water-solubility. Contradictory, particle size distributions of carbon microspheres from native starch tended to broaden distributions that confirm-ed from SEM observations.

The energy-dispersive X-ray (EDX) analyses of carbon microspheres from various types of starch after carbonization process are shown in Table 1. These results showed that carbon is the main component of the CMSs in the range of 66-71wt%. However, the oxygen component in carbon microspheres might mainly come from the absorbed water molecules in pore structure [18].

In addition, the XRD patterns of some CMSs after carbonization are shown in Figure 3. There are the presences of two broad peaks at 2 = 24.8 and 43.5 which are reflections from the (002) plane and the (101) plane, respectively. The peaks can be indexed to a hexagonal graphite lattice. The broadening of the peaks suggests the presence of an amorphous carbon phase within the CMSs [19]. These semi-hexagonal graphite struct-ures took place during carbonization process at high temperature. In carbonization process, carbon atom in CMSs would be rearranged to form graphene sheets and partially collapsed to yield pore structures. This structure would be suitable for any substances storage which have their molecule sizes less than micropore (< 2 nm).

All N2 adsorption-desorption isotherms of CMSs after carbonization process were shown in Figure 4. These isotherms indicated that the carbonized CMSs exhibited type I adsorption isotherm due to its micropore structure regarding to IUPAC classification [20]. The Brunauer–Emmett–Teller (BET) surface areas of CMSs before and after carbonization were also summarized in Table 2. The CMSs surface areas were dramatically increased after carbonization process. The release of H, O and C during carbonization process increased large quantities of micro-pores throughout the bulk of the samples [7].

Figure 2. Particle size distribution of CMSs after carbonization which are ()HICAP-®100, ()CAPSUL, ()Tapioca, ()Corn, ()Rice, ()Wheat, ()Sticky rice.

Table 1. The elemental components of CMSs from energy dispersive X-ray.

Elemental components Types of starches Carbon (%) Oxygen (%)

HICAP®100 71.08 28.92 CAPSUL® 68.15 31.85 Tapioca 69.11 30.89 Corn 66.67 33.33 Rice 68.44 31.56 Wheat 67.72 32.28 Sticky rice 69.01 30.99

Figure 3. X-ray diffraction patterns of CMSs after carbonization at 600 oC.



44

4. Conclusions

High micropores and monodisperse CMSs were synthesized via a facile hydrothermal process without any catalysts. The low-cost starting materials and moderate reaction temperature provide an efficient way to fabricate solid CMSs. Furthermore, the particle size distribution of the CMSs strongly depended on types of starch. In other words, the smallest particle size of CMSs could be synthesized from HICAP®100 because it can be hydrolyzed immediately to form nucleates of CMSs. On the other hand, broaden particle size distributions of CMSs from some types of native starch because they were continuously hydrolyzed during hydrothermal process to grow shell of CMSs. This mechanism came from their water-insolubility of native starch. Furthermore, high porosity of CMSs could be developed by treating the dried CMSs in carbonization process. The BET surface area of CMSs after carbonization process dramatically increased from 1-5 m2/g to 400-500 m2/g. In addition, the CMSs also increased their carbon contents by losing oxygen and hydrogen atoms during carbonization process. There-fore, the rearrangement of carbon atom in CMSs to form grapheme sheets took place.

Acknowledgements

This work was financially supported by the Centennial Fund of Chulalongkorn University for Center of Excellence in Particle Technology (CEPT) and Department of Chemical Engineering, Faculty of Engi-neering, Chulalongkorn University.

References

[1] Kroto, H.W., Heath, J.R., O’Brien, S.C., Curl, R.F., & Smalley, R.E. (1985). C60: Buck-minsterfullerene. Nature, 318(6042), 162-3.

[2] Iijima, S. (1991). Helical microtubules of graphitic carbon. Nature, 354(6348), 56-8.

[3] Sun, X., & Li., Y. (2004). Colloidal carbon spheres and their core/shell structures with noble-metal nanoparticles. Angewandte Chemie-International Edition, 43(5), 597-601.

Figure 4. N2 adsorption-desorption isotherm of CMSs after carbonization which are ()HI-CAP®100, ()CAPSUL, ()Tapioca, ()Corn, ()Rice, ()Wheat, ()Sticky rice.

Table 2. Specific BET surface area of CMSs.

Specific BET surface area, SBET [m2/g] Types of

starches Before carbonization

After carbonization

HI-CAP®100

4.32 560

CAPSUL® 3.41 530 Tapioca 3.23 546 Corn 3.57 520 Rice 2.89 457 Wheat 1.23 444 Sticky rice 3.12 415



45

[4] Jang, J., & Bae, J. (2004). Fabrication of polymer nanofibers and carbon nanofibers by using a salt-assisted microemulsion polymer-ization. Angewandte Chemie-International Edition, 43(29), 3803-6.

[5] Yuan, D., Xu, C., Liu, Y., Tan, S., Wang, X., Wei, Z., & Shen, P.K. (2007). Synthesis of coin-like hollow carbon and performance as Pd catalyst support for methanol electro-oxidation. Electrochemistry Communicat-ions, 9(10), 2473-8.

[6] Xiao, Y., Liu, Y. , Cheng, L., Yuan, D., Zhang, J., Gu, Y., & Sun, G. (2006). Flower-like carbon materials prepared via a simple solvothermal route. Carbon, 44(8), 1589-91.

[7] Serp, P., Feurer, R., Kalck, P., Kihn, Y., Faria, J.L., & Figueiredo, J.L. (2001). Chemical vapour deposition process for the production of carbon nanospheres. Carbon, 39(4), 621-6.

[8] Auer, E., Freund, A. (1998). Carbons as supports for industrial precious metal catalysts. Applied Catalysis A: General, 173(2): 259-71.

[9] Vignal, V., Morawski, A.W., Konno, H., & Inagaki, M. (1999). Quantitative assessment of pores in oxidized carbon spheres using scanning tunneling microscopy. Journal of Materials Research, 14(3), 1102-12.

[10] Lou, Z., Chen, Q., Gao, J., & Zhang, Y. (2004). Preparation of carbon spheres consisting of amorphous carbon cores and graphene shells. Carbon, 42(1), 229-32.

[11] Inagaki, M., Vignal, V. (1999). Effects of carbonization atmosphere and subsequent oxidation on pore structure of carbon spheres observed by scanning tunneling microscopy. Journal of Materials Research, 14(7), 3152-7.

[12] Yang, R., Li, H., Qui, X., & Chen, L. (2006). A spontaneous combustion reaction for synthesizing Pt hollow capsules using colloidal carbon spheres as templates. Chemistry-A European Journal, 12(15), 4083-90.

[13] Mi, Y., Hu, W., Dan, Y., & Liu, Y. (2008). Synthesis of carbon micro-spheres by a glucose hydrothermal method. Materials Letters, 62(8-9), 1194-6.

[14] Yuan, D., Xu, C., Liu, Y., Tan, S., Wang, X., Wei, Z., & Shen, P.K. (2007). Synthesis of coin-like hollow carbon and performance as Pd catalyst support for methanol electro-oxidation. Electrochemistry Communicat-ions, 9(10), 2473-8.

[15] Tusi, M.M., Brandalise, M., Correa, O.V., Neto, A.O., Linardi, M., & Spinace, E.V. (2009). Preparation of PtRu/C electrocatal-ysts by hydrothermal carbonization process for m ethanol electro-oxidation. Portugaliae Electrochimica Acta, 27(3), 345-52.

[16] Sun, X., Li, Y. (2004). Colloidal carbon spheres and their core/shell structures with noble-metal. Nanoparticles. Angewandte Chemie-International Edition, 43(5), 597-601.

[17] Yang, R., & Qiu, X. (2005). Monodispersed hard carbon spherules as a catalyst support for the electrooxidation of methanol. Carbon, 43(1), 11-6.

[18] Mi, Y., Hu, W., Dan, Y., & Liu, Y. (2008). Synthesis of carbon microspheres by a glucose hydrothermal method. Materials Letters, 62(8-9), 1194-6.

[19] Zheng, M., Liu, Y., Xiao, Y., Zhu, Y., Guan, Q., Yuan, D., & Zhang, J. (2009). An easy catalyst-free hydrothermal method to prepare monodisperse carbon microspheres on a large scale. Journal of Physical Chemistry C, 113(19), 8455-9.

[20] Sing, K.S.W., & Gregg, S.J. (1982). Adsorption, surface area and porosity. (2), New York: Academic Press.

[21] Wang, Q., Li, H., Chen, L., & Huang, X. (2001). Monodispersed hard carbon sphe-rules with uniform nanopores. Carbon, 39(14), 2211-4.




Research Article

Immobilization of Lipase on CaCO3 and Entrapment in Calcium Alginate Bead for Biodiesel Production

N. Sawangpanya, C. Muangchim, M. Phisalaphong*

Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.


1. Introduction

Due to the limitation of fossil fuels, biodiesel becomes an attractive alternative energy. Biodiesel is a renewable energy. It is clean and environmentally safe. Biodiesel can be obtained from vegetable oils by trans-esterification with short chain alcohols to form esters. Chemical catalysts are widely developed to improve the reaction rate. Nevertheless, there are several drawbacks

from using alkali or acid catalyst processes, for example; high energy requirements, diffi-culties in the recovery of catalyst and glycerol and pollution from waste water. The biocatalyst such as lipase can eliminate the drawbacks of chemical catalysts by producing product of very high purity and offers an environmentally attractive option [1-4]. However, the hurdle to use of lipase for biodiesel fuel production is the cost of biocatalyst. The use of immobilized enzymes could overcome this problem. Immobiliz-ations of lipase can be achieved by adsorption onto support matrices such as particles,

Abstract

Biodiesel productions from palm oil by using C. rugosa free and immobilized lipases as biocatalysts were investigated. Three methods of lipase immobilizations were studied: 1) adsorption of lipase on CaCO3 (CRLA), 2) entrapment of lipase in Ca-alginate matrix (CRLE) and 3) entrapment of CaCO3–lipase in Ca-alginate matrix (CRLAE). The effects of operational parameters such as ratio of enzyme to oil, molar ratio of ethanol to oil, operating temperature, bead diameter and shaking speed on transesterification were examined. The optimal conditions for the biodiesel were at the molar ratio of ethanol to palm oil of 9:1, 5 % C. rugosa lipase based on oil weight, 50 oC and 250 rpm. After 48 hours at the optimal condition, the reaction with entrapment of lipase of Ca-alginate matrix (CRLE) exhibited the higher ethyl ester yield (74%) than those of CRLA and CRLAE, whereas ethyl ester yield of 83% was obtained after 24 hours by using the free lipases.

Keywords: Alginate, Biodiesel, Calcium carbonate, Immobilization, Lipase.

N. Sawangpanya et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 46-51


47

fibers, by entrapping them in gel matrices or by covalent attachment [1]. The enzyme immobilization by adsorption onto a solid support or entrapment in bio-polymer matrix such as calcium alginate remains the most simple and cost-effective. It was suggested that the immobilized lipase facilitates mass transfer by spreading the enzyme on a large surface area and by preventing the enzyme particles from aggregation [4]. The CaCO3 presented the advantages of being non-toxic and lacking of chemical reactivity. Further-more, this support was selected as a suitable adsorbent leading to high dispersion of the crude R. oryzae lipase in the support and preserving the catalytic activity [2]. Its readsorption is sometimes possible by modification of the pH, followed by binding of new active enzyme [4]. However, ready desorption would also be a major drawback of this immobilization technique if it occurs during the catalyzed reaction. The other physical immobilization of a lipase is its inclusion in an insoluble polymer or entrap-ment in bio-polymer matrix such as calcium alginate is attractive. The advantage of such an immobilization technique is that the enzyme does not chemically interact with the polymer; therefore, denaturation is possibly avoided [5].

This study exploits the idea on developing a new enzyme carrier by adsorption of lipase on CaCO3 followed by the entrapment in calcium alginate beads (CRLAE) for appli-cation in biodiesel production using purified palm oil and 95% (v/v) ethanol as substrates. The result was compared with those using free enzymes, immobilized enzymes in con-ventional calcium alginate beads (CRLE) and enzymes immobilized by adsorption on CaCO3 (CRLA).


Enzyme and Chemicals. Candida rugosa lipase (EC 3.1.1.3) was received as a gift sample from Amano Pharmaceuticals, Japan. Virgin olive oil was purchased from local market. Carbonate of calcium (CaCO3) was

from Univar. All substances are the analytical grade. The enzyme immobilization was made on to CaCO3 according to Rosu et al. (1997) with a slight modification. A support powder (2 g) was added to 20 ml of enzymatic solution. The mixture was incubated 1 h at 4 ºC under mild agitation. Afterwards, 10 ml of chilled acetone was added, and the suspension was filtered through a Buchner funnel, the preparation of immobilized lipase was washed two times with another 100 ml aliquot of chilled acetone, dried in vacuum desiccators at room temperature for 1-2 h and stored at 4ºC until use.

Entrapment of C. rugosa Lipase Immobilized on CaCO3 in Calcium Alginate Beads (CRLAE). C. rugosa lipase which pre-immobilized on CaCO3 by adsorption was entrapped in calcium alginate beads. The sodium alginate solution of 1%w/v and 0.1 M calcium chloride solution was prepared in 0.01 M sodium phosphate buffer (pH 7.0). The immobilized enzyme on CaCO3 was dispersed uniformly in 1% sodium alginate solution and was injected through a syringe into 0.1 M calcium chloride solution from a constant distance. The beads were allowed to harden in calcium chloride solution for an hour.

Lipase Hydrolysis Activity. The hydrolysis activity was assayed titrimetrically using olive oil emulsion method [7]. The substrate was prepared by mixing 50 ml of olive oil with 50 ml of Arabic gum solution (7%w/v). The reaction mixture consisting of 20 ml of emulsion, 2 ml of 0.1 M sodium phosphate buffer, pH 7.0 and immobilized lipase (200 mg) was incubated for 12 h at 37 °C. The liberated fatty acid was titrated with 0.05 N potassium hydroxide solution using Phenol-phthalein as an indicator. One unit (U) of enzyme activity was defined as the amount of enzyme that produced 1 μmol of free fatty acids per min under the assay conditions.




48

Biodiesel production from purified palm oil and 95% (v/v) ethanol by using C. rugosa in forms of free and immobilized lipases as biocatalysts was studied. The influence of operating conditions on enzyme activity was investigated using lipase-CaCO3 immobilized in calcium alginate beads (CRLAE) in comparison to that using free enzyme, and immobilized enzymes adsorbed on CaCO3 (CRLA) and entrapped in calcium alginate bead (CRLE).

Effect of Lipase Quantity. The effect of lipase quantity on the alcoholysis of purified palm oil was investigated by varying free lipase quantities at 1%, 3%, 5% and 10 % based on palm oil weight with a reaction temperature of 50°C. The results are presented in Figures 1 and 2. The hydrolytic activity and ethyl ester content were increased by increasing lipase quantity up to 5%. The highest conversion was obtained when 5-10 % C. rugosa lipase based on oil weight was used. The maximum hydrolytic activity and ethyl ester yield were about 120 U and 83.4 %, respectively. According to Kose et al. (2001), the highest ethyl ester formation (82.6%) was obtained by using 30% C. antarctica lipase based on cotton seed oil weight with an operation temperature at 50°C. Awang et al. (2007) reported the highest conversion of 79.5% when 10% (w/w) of C. rugosa lipase concentration was used for esterification of oleic acid and oleyl alcohol in hexane. From the result of this study, C. rugosa lipase at 5% (by wt. of oil) was applied for the further study.

Effect of Temperature. The effect of temper-ature on the activity of free enzymes and immobilized enzymes was investigated by using olive oil as a substrate at pH 7.0 in the temperature range of 37-60 °C as shown in Figure 3. The activity of the lipase increased with the increase of the operating temperature up to 50°C. The maximum activity of both free and immobilized enzymes appeared at 50°C. However, above 50°C, the enzyme activity of the free enzyme was significantly decreased with the increasing temperature, while the enzyme activity of the immobilized

enzymes slightly decreased. Therefore, the immobilization in CRLE, CRLA and CRLAE carriers could provide good heat resistance.

0

30

60

90

120

150

0 2 4 6 8 10 12

lipase quantity (wt% of oil)

Hyd

roly

tic

acti

vity

(U

)

Figure 1. Effect of C. rugosa lipase quantity on hydrolytic activity, at pH 7.0, 50°C, 250 rpm and reaction time of 12 h.

0.00

20.00

40.00

60.00

80.00

100.00

1 3 5 10

Lipase quantity (wt% of oil)

Eth

yl E

ster

Yie

ld (

%)

Figure 2. Effect of C. rugosa lipase quantity on ethyl ester yield with molar ratio of ethanol to palm oil 9:1, reaction temperature of 50°C, 250 rpm and reaction time of 24 h.

Effect of Molar Ratio of Ethanol to Palm Oil. The effect of molar ratio of ethanol to palm oil on ethyl ester yield was determined under reaction temperature of 50°C. The ethyl ester yield in Figure 4 was obtained after the reaction was carried out for 24 h. The ethyl ester yield increased from about 47% to 83 % as the molar ratio increased from 3:1 to 9:1 and considerably decreased after that. In the enzymatic catalysis in aqueous medium, the nature of the organic solvent influences the activity and the stability of the enzymes considerably. Highly polar and hydrophilic solvents are capable of solubilizing large



49

amounts of water. The removing of essential water from the enzymes could cause significant loss of the catalytic activity [10]. The result demonstrated the optimal molar ratio of ethanol to reactants for catalytic transesterification of palm oil at 9:1.

0

30

60

90

120

150

20 30 40 50 60 70

Temperature (C)

Hyd

roly

tic

activi

ty (

U)

Figure 3. Effect of temperature on the hydrolytic activity of free and immobilized C. rugosa lipases at pH 7.0 and 12 h; ♦, free lipase; *, CRLA; ∆, CRLE; □, CRLAE.

Figure 4. Effect of molar ratio of ethanol to palm oil on ethyl ester yield by using free lipase at 5% by wt of oil, reaction temperature of 50°C, 250 rpm and reaction time of 24 h.

Effect of Shaking Speed. Figure 5 presents the effect of shaking speed on soluble lipase mediated transesterification of purified palm oil at 50˚C, ethanol:oil molar ratio at 9:1 with using 5% free lipase (by wt of oil). It was found that the final conversion yield was

accelerated with the increase of shaking speed up to about 250 rpm. The external mass transfer limitation was not so significant when the rotating speed was greater than 250 rpm. At 250 rpm, the final ethyl ester yield of 79.6 % was obtained, which was only slightly less than that of shaking speed at 300 rpm (81.9%). Although some minor variation from the result of the previous study was observed, the result of this study clearly demonstrated the rise of reaction rate with increasing shaking speed up to 250 rpm. It could be explained that at high shaking speed, the reaction system was thoroughly emulsified and the interfacial area was increased evidently. Consequently, the react-ion was facilitated because the collision probability between lipase and substrate was improved greatly [11].

0

20

40

60

80

100

0 4 8 12 16 20 24

Time (hr)

Eth

yl E

ster

Yie

ld (

%)

Figure 5. Effect of shaking speed on ethyl ester yield by using the free lipases (5% based on oil weight). The molar ratio of ethanol to palm oil was 9:1 at the reaction temperature of 50°C, 24 h; ●, 150; ∆, 200; ■, 250; *, 300.

Effect of Diameter of Immobilized Bead. The effect of bead diameter on the activity of immobilized lipase was investigated by varying the diameter of bead at 1.7, 2 and 4 mm with 1% sodium (Na)-alginate concentration (Figure 6). The activity of CRLAE was significantly lower than that of CRLE. The Na-alginate concentration and bead diameter for the maximum activity (184.17 U) were 1% w/v and 1.7 mm, respectively. Na-alginate ≥ 1.5 % w/v could cause the strong

0.00

20.00

40.00

60.00

80.00

100.00

1 2 3 4 5

(a)

Eth

yl e

ster

yie

ld (

%)

3:1 6:1 9:1 12:1 15:1



50

limitation in internal diffusion of the bead (data not shown). It was found that the activity of immobilized lipase was decreased with the increase of bead diameter. The diffusion interference in hydrolytic reaction over the calcium alginate bead intruded to slow the rate of reaction. Moreover, it was found that the lack of buffer solution in the bead could be the cause of the lower lipase activity.

0

50

100

150

200

0 1 2 3 4

Diameter of alginte beads (mm)

Hyd

roly

tic

acti

vity

(U

)

Figure 6. Effect of bead diameter on the hydrolytic activity of immobilized C. rugosa lipase with 1% Na-alginate concentration; (), CRLE carrier; (▲), CRLAE carrier with buffer solution; (), CRLAE carrier prepared without buffer solution.

Ethyl Ester Yields of Biodiesel by Catalytic Transesterification. The optimum reaction conditions from the previous study were employed for transesterification using immo-bilized lipase. The ethyl ester yields of biodiesel by catalytic transesterification of purified palm oil are shown in Figure 7. The ethyl ester yield by using the immobilized lipase was lower than that of the free lipase (83.4%). After 48 hours, the entrapment of C. rugosa lipase in Ca-alginate matrix (CRLE) showed the higher ethyl ester yield (74.2 %) than that of the immobilization of C. rugosa lipase adsorbed on CaCO3 (CRLA) (57.6 %)

and CaCO3-lipase entrapped in Ca-alginate (CRLAE) (42.7%), respectively.

0.00

20.00

40.00

60.00

80.00

100.00

0 6 12 18 24 30 36 42 48

Time (hr)

Eth

yl E

ster

Yie

ld (

%)

Figure 7. The ethyl ester yield by using the different techniques of immobilized lipase.♦, free lipase; ○, CRLE;▲, CRLA; □, CRLAE; *, CaCO3.

4. Conclusions

Biodiesel productions from purified palm oil and 95 % (v/v) ethanol by using C. rugosa- free and immobilized lipases as biocatalysts were investigated. The optimal conditions were at the molar ratio of ethanol to palm oil of 9:1 using 5% wt (by oil) lipase, controlled at temperature 50 °C, 250 rpm and reaction time 24 h, with the yields of ethyl ester at 83.4% whereas the biodiesel production by the immobilized lipase in CRLE, CRLA and CRLAE resulted in ethyl ester yields of about 74.2, 57.6 and 42.7, respectively after 48 hours at the optimal condition.

Acknowledgements

This work was financially supported by the Thailand Research Fund (TRF) and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) under grant number MRG-WII 515E011.



51

References

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[7] Yadav, G.D., & Jadhav, S.R. (2005). Syn- thesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterifica- tion in non-aqueous medium. Microporous and Mesoporous Mat. 86, 215-22. [8] Kose, O., Tuter, M., & A yse Aksoy, H. (2002). Immobilized Candida Antarctica lipase-catalyzed alcoholysis of cotton seed oil in a solvent-free medium. Bioresource Technology, 83, 125–9. [9] Awang, R., Ghazuli Rafaei, M., & Baari, M. (2007). Immobilization of lipase from Candida rugosa on palm-based polyurethane foam as a support material. American Journal of Biochemistry and Biotechnology, 163-6. [10] de A. Vieire, A.P., da Silva, M.A.P., & Langone, M.A.P. (2006). Biodiesel production via esterification reactions catalyzed by lipase. Latin American Applied Research, 36, 283-8. [11] Xin, C., Wei, D., & Liu, D. (2008). Effect of several factors on soluble lipase mediated biodiesel preparation in the biphasic aqueous -oil systems. J. Microbiol Biotechnol, 24, 2097-102.




Research Article

Phytoremediation : Vetiver Grass in Remediation of Soil Contaminated with Trichloroethylene

J. Janngam1, P. Anurakpongsatorn 1*, T. Satapanajaru1, S. Techapinyawat 2

1Department of Environmental Science, Faculty of Science, Kasetsart University, Jatuchak, Bangkok 10900, Thailand.

2Department of Botany, Faculty of Science, Kasetsart University, Jatuchak, Bangkok 10900, Thailand.

1. Introduction

Phytoremediation is an emerging green technology that uses plants to remediate soil, sediment, surface water, and groundwater environments contaminated with toxic met-als, organics, and radionucides [4]. This method has the benefit of contributing to site restoration when remedial action is

ongoing. The action of plants can include the degradation, adsorption, accumulation and volatilization of compounds or the enhancement of soil rhizosphere activity. Many different compounds and classes of compounds can be removed from the environment by this method, including solvents in groundwater, petroleum and aromatic compounds in soils, and volatile compounds in the air [6]. Phytoremediation is more cost-effective than alternative mechani-cal or chemical methods of removing hazard-ous compounds from the soil [4].

Abstract

Trichloroethylene (TCE) is chlorinated hydrocarbon which used in degreasing oil and grease from process products. It was found that this chemical was contaminated in environmental, soil and water around industrial area. Soil was collected from Pratum Thani province which had TCE higher than the standard set by Ministry of Industry in Thailand. Four ecotypes of vetiver grass (Vetiveria zizanioides) were used for phytoremediation including Songkla3, Sri Lanka, Kamphaeng Phet2 and Surat Thani. All ecotypes grew up and the survival rate was 100% after planting for 1 month. Surat Thani had the most number of leaves (6.67±0.58). Songkhlar3 had the longest shoots followed by Sri Lanka (6.67±0.29, 5.33±0.76 cm). Songkla3 and Sri Lanka had the longest leaves (40.57±1.39 and 39.30±5.88 cm). However, there were no statistical differences (p > 0.05) in sprouts quantity and leaves width among the four ecotypes. Songkhlar3 and Sri Lanka were selected for further experiment. Vetiver grass was planted in contaminated soil mixed with soil conditioners including coconut residue : soil : manure in ratio 3 : 2 : 1 by weight. TCE was higher accumulated in leaves than shoots and roots. The removal of TCE from contaminated soil was about 98% for two ecotypes. However, in the field trial more parameters would be put to concern than laboratory scale.

Keywords: Phytoremediation, Trichloroethylene, Vetiver grass.

J. Janngam et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 52-57


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Trichloroethylene (TCE) is one of the most common environmental pollutants in the industrialized world. It has been used for decades in military, industrial, medical and household settings, in a wide range of applications including uses as a metal degreaser, dry cleaning agent, and anaesthe-tic. TCE is very stable, and in some aquifers it has persisted for decades. It is known to be a hepatotoxin, and there is also growing evidence that it may be a carcinogen [13]. In Thailand, TCE was found contaminated in soil and groundwater beside industries [9]. Many recent researches have shown that TCE can be taken up by plants in both lab scale experiments and field-site-scale studies [15] for example, tobacco, poplar [7,13], carrots, spinach, tomatoes [14], cattail, eastern cot-tonwood [5], apple and peach tree [2]. To find another plant and can grow in a TCE contaminated site that has the efficiency to take up TCE from soil, we selected vetiver grass (Vetiveria zizanioides) to determine for the capability to remediate contaminated soil. by phytoremediation.

Vetiver grass is a perennial grass belonging to the Poaceae family. It has short rhizomes and a massive, finely structured root system. The deep root system makes the vetiver grass extremely drought tolerant and very difficult to dislodge when exposed to a strong water flow. Likewise, the vetiver grass is also highly resistant to pests, diseases, fire [8]. It is known to be tolerant to heavy metals [10]. There are reports on the use of this plant for phytoremediation of soils contaminated with heavy metals [3,16], phenol [11], radionuclides and nuclear waste [12]. However, there was no report about using vetiver grass for phytoremediation of soil contaminated with TCE. The goal of this report is to examine four ecotypes of vetiver grass including, Songkla3, Sri Lanka, Kamphaeng Phet2 and Surat Thani for their ability to grow in soil contaminated with TCE and to investigate phytoremediation efficiency of Songkla3 and Sri Lanka. The result was used to consider

the potential to use vetiver grass to remediate TCE contaminated area.


All of the vetiver grass plantlets were received from Office of The Royal Develop-ment Projects and the research site was located in the Department of Environmental Science, Faculty of Science, Kasetsart Uni-versity.

Plant Material. The plantlets of vetiver grass (Vetiveria zizanioides) for four ecotypes including Songkla3, Sri Lanka, Kamphaeng Phet2 and Surat Thani were used for the experiments. To compare the growth capabi-lity of vetiver grass in TCE contaminated soil, we used four ecotypes of them. The age of the plants was between 1-1.5 months and all of them were planted in planting material to develop root system for 3 weeks. They were maintained and cut for 25 cm length before planted in soil contaminated with TCE (1 plant/pot).

Another experiment analyzed for the accu-mulation of TCE in plant parts, two ecotypes were selected. The plantlets were prepared as same as the first experiment but they were planted in soil mixed with soil conditioners. The plant pots were put in clear-roof, opened building. Water was applied every 2 days.

Soil Preparation. Soil was collected from Pratum Thani province in Thailand. The soil was collected from a lagoon. The character-istic of the soil were clay. The soil was air-dried, crushed to pass through a 1 cm dia-meter sieve, and mixed thoroughly. Analyzed for texture, moisture after air dried, pH, organic matter (OM), carbon exchange capa-city (CEC) and TCE concentration. The prepared soil was separated into 2 conditions. First, put into pots (5 kg/pot) for testing the growth capability of vetiver grass in this soil. Second, mixed with soil condit-ioners including coconut residue : soil : animal manure in a ratio 3 : 2 : 1 by weight as

Phytoremediation : Vetiver grass in Remediation of Soil contaminated with Trichloroethylene


54

a common ratio for planting material. One kilogram of mixture was added in each pot.

Samples Analysis. Soil samples were collect-ed and 5 g of the soil was put into a 25 ml amber vial. All samples were extracted with 5 ml methanol and 25 ml deionized water, closed immediately with Teflon line rubber and aluminium cap and kept at room temper-ature for 24 h. Plant samples were prepared by cutting and dividing into root, shoots and leaves, 5 g of samples were extracted with 5 ml conc. H2SO4 and 15 ml deionized water, closed immediately with Teflon line rubber and aluminium cap. After that, heated with a hot plate at 90°C for 30 min and kept at room temperature for 48 h. All soil and plant samples were shaken with incubator shaker at 25°C, 60 rpm. Head space sampler HP 7694 was used for shaking for 10 min. TCE was analyzed using a Gas chromatography electron capture detector (GC-ECD) HP 6890 series using a capillary (HP-5) 30 m x ID 0.32 mm, film thickness 0.25 µm coated with 5% phenyl-methylpoly-siloxane. Column inlet temperature 210°C, oven temperature 90°C, carrier gas flow 1.50 ml/min with velocity 34 cm/sec.

Translocation in the plant parts from shoot to root was measured using Translocation factor (TF) which is given below Eq. (1) [1].

TF = Cs/Cr (1)

Where, Cs and Cr are TCE concentrations (mg/kg) in the shoot and root, respectively. Wherein, TF > 1 indicates that the plant has an efficiency to translocate TCE from root to shoot.

Experimental Design. All four ecotypes of vetiver grass were cut to 15 cm height and planted in the soil collected from Pratum Thani province in Thailand (5 kg/pot). The lay out plan was completely randomized design with 3 replications, outdoors grown

with no chemical fertilizers. After 1 month, the number of leaves, length of leaves, length of shoot, width of leaves and quantity of sprouts were measured. Two ecotypes that had the best efficiency growth were selected for the further experiment.

Selected vetivier grasses (2 ecotypes) were used in planting in soil mixed with soil conditioners. They were planted for 1 month and the TCE was measured in plant parts and planting materials. This experiment was set up to check the TCE accumulation in plants. SPSS program was used with 0.05 significant difference.


Characteristic of Soil. The soil was extreme-ly acidic (pH3.8), organic matter (OM) 2.11%. It was high in carbon exchange capacity (CEC) 22.37 mol/kg and the texture was clay (clay = 55.60%). Because of the high value of clay content, made it difficult for water to pass through the soil. Moisture after air drying was 5.29%. TCE concentra-tion was 549 mg/kg, which was higher than the standard set by Ministry of Industry in Thailand; 28 mg/kg (Table 1).

Table 1. Characteristic of soil.

Parameter Value

Texture Clay Soil moisture 5.29% pH 3.8 OM 2.11% CEC 22.37 mol/kg TCE 549 mg/kg

Growth Capability of Vetiver Grass. It was found that all ecotypes of vetiver grass can grow in TCE contaminated soil and had a 100% survival. The statistical result of growth capability at significance 0.05 levels showed that Surat Thani had the largest number of leaves (6.67±0.58) different from the other ecotypes (Figure 1). Sri Lanka, Songkhlar3 and Kamphaeng Phet had similar



55

number of leaves, 5.33±0.58, 5.00±0.00, and 5.00±0.00, respectively.

5.335.00 5.00

6.67

0

1

2

3

4

5

6

7

8

Surat Thani Sri Lanka Songkhlar3 KamphaengPhet2

Nu

mb

er o

f le

aves

Surat Thani Sri Lanka Songkhlar3 Kamphaeng Phet2

Figure 1. Number of leaves, planting in TCE contaminated soil.

6.67

5.33 5.07 4.9

0

1

2

3

4

5

6

7

8

Songkhlar3 Sri Lanka Surat Thani KamphaengPhet2

Shoo

ts le

ngth

(cm

)

Songkhlar3 Sri Lanka Surat Thani Kamphaeng Phet2

Figure 2. Shoots length, planting in TCE contaminated soil. Songkhlar3 had higher shoot length followed by Sri Lanka and Surat Thani (6.67±0.29, 5.33±0.76 and 5.07±1.21 cm). The statistical result at significance 0.05 levels showed Kamphaeng Phet2 had the shortest of shoot length (4.90±0.96 cm) different from other ecotypes (Figure 2). The result of shoot length was in the same trend of leaves length. Songkhlar3 had the most leaves length followed by Sri Lanka, Kamphaeng Phet2 and Surat Thani (40.57±1.39, 39.30±5.88, 32.48±2.67 and 26.21±4.64 cm, respectively) (Figure 3). However, there was no statistical differences (p > 0.05) in sprouts quantity and leaves width among the 4 ecotypes (Table 2). Kamphaeng Phet2, Songkhlar3, Surat Thani and Sri Lanka had similarly number of sprouts 4.00±1.00, 2.67±1.53, 2.67±0.58 and 2.00±1.00 cm and the leaves width

were 0.76±0.09, 0.77±0.05, 0.77±0.02 and 0.74±0.99 cm, respectively. From this experi-ment, two ecotypes that gave the best result in growth capability were selected. Thus, we selected Songkhlar3 and Sri Lanka for further experiment. The difference in the growth of four ecotypes may due to both of the growing nature of the plants and the efficiency to tolerate TCE in soil.

40.57 39.30

32.48

26.21

0

5

10

15

20

25

30

35

40

45

Songkhlar3 Sri Lanka KamphaengPhet2

Surat Thani

Lea

ves

leng

th (

cm)

Songkhlar3 Sri Lanka Kamphaeng Phet2 Surat Thani

Figure 3. Leaves length, planting in TCE contaminated soil.

TCE Accumulation in Plant Parts. The result showed that TCE was accumulated in leaves more than root and shoots (Table 3). Sri Lanka had more TCE accumulated in leaves than Songkhlar3 (4.48±1.07 and 4.06±2.18 mg/kg) and accumulation in shoots were 4.04±2.44 and 3.71±1.04 mg/kg. However, in root Songkhlar3 had more TCE accumul-ation than Sri Lanka (2.73±1.36 and 1.74±0.42). It is the advantage of vetiver grass that it’s plant parts above ground level had capability to accumulated TCE. 1) TCE was taken out from the soil via harvesting of the above ground level parts. 2) While vetiver grass was growing, TCE was translocated from soil to root to new shoots and leaves. That left the soil cleaner.

TF of TCE in two ecotypes of vetiver grass was higher than 1 (Table 4). It was indicated that vetiver grass had an efficiency to translocate TCE from root to shoot. TCE was analyzed from soil left in the pots. The result showed that TCE removal was very high (Table 4). About 98% of TCE was lost after growing vetiver grass for 1 month, Song-khlar3 and Sri Lanka showed the same trend (98.39% and 98.36%, respectively).

Phytoremediation : Vetiver grass in Remediation of Soil contaminated with Trichloroethylene


56

Table 2. Growth capability of vetiver grass.

Ecotypes Number of Leaves

Shoot Lngth (cm)

Leaves Length (cm)

Number of Sprouts

Leaves Width (cm)

K2 5.00±0.00 b 4.90±0.96 b 32.48±2.67 bc 4.00±1.00 a 0.76±0.09 a Si 5.33±0.58 b 5.33±0.76 ab 39.30±5.88 ab 2.00±1.00 a 0.74±0.09 a So3 5.00±0.00 b 6.67±0.29 a 40.57±1.39 a 2.67±1.53 a 0.77±0.05 a Su 6.67±0.58 a 5.07±1.21 ab 26.21±4.64 ab 2.67±0.58 a 0.77±0.02 a

Note: Mean and standard deviation (n=3); K2=Kamphaeng Phet; Si=Sri Lanka; So3=Songkhlar3; Su=Surat Thani; small letters stand for significance at 0.05 levels

Table 3. TCE concentration in plant parts (mg/kg).

TCE concentration (mg/kg) Plant Parts Sri Lanka Songkhlar3 Root 1.74±0.42 b 2.73±1.36 a Shoots 4.04±2.44 ab 3.71±1.04 a Leaves 4.48±1.07 a 4.06±2.18 a

Note: Mean and standard deviation (n=4); small letters stand for significance at 0.05 levels

Table 4. Translocation factor (TF) and %removal of TCE in soil mixed with soil conditioners.

Treatment TF %Removal Sri Lanka 2.32 98.36 Songkhlar3 1.36 98.39

4. Conclusions

Four ecotypes of vetiver grass including, Songkla3, Sri Lanka, Kamphaeng Phet2 and Surat Thani, were planted in soil contam-inated with TCE for 1 month. All of them had 100% of survival rate. Songkhlar3 and Sri Lanka showed the best growth capability. These two ecotypes were selected to plant in soil mixed with soil conditioners including

coconut residue : soil : animal manure at ratio 3 : 2 : 1 by weight to determine for the TCE accumulation in plant parts. The results showed that TCE was removed from this mixture materials and accumulated mostly in leaves followed by shoots and root. Wherein, TF > 1 indicated that TCE had an efficiency to translocate from root to shoots. TCE removal from soil was very high about 98% of TCE was lost from soil mixed with soil conditioners. This is an advantage for this plant for phytoremediation. While, TCE was accumulated in leaves, cutting leaves could remove TCE from contaminated soil. Vetiver grass was then left to grow in site to continuous removing TCE. However, more parameters would be put in considerations in the field site compared to laboratory scale study.

Acknowledgements

Authors thank Faculty of Science, Kasetsart University and The Thailand Research Fund (TRF) for their support with funding. Thankful to Office of The Royal Development Projects and Reference Laboratory and Toxicology Center.

References [1] Bu-Olayan, A.H., & Thomas, B.V. (2009).

Translocation and bioaccumulation of trace metals in desert plants of Kuwait govern-

orates. Research Journal of Environmental Sciences 3. 5, 581-7.

[2] Chard, B.K., Doucette, W.J., Chard, J.K.,



57

Bugbee, B., & Gorder, K. (2006). Trichloro-ethylene uptake by apple and peach trees and transfer to fruit. Environ. Sci. Technol., 40, 4788-93.

[3] Wilde, E.W., Brigmon, R.L., Dunn, D.L., Heikamp, M.A., & Dagnan, D.C. (2005). Phytoextraction of lead from firing range soil by Vetiver grass. Chemosphere., 61, 1451-7.

[4] Alkorta, I., & Garbisu, C. (2001). Phytore-mediation of organic contaminants in soils. Bioresource Technology., 79, 273-6.

[5] Bankstona, J.L., Solab, D.L., Komora, A.T., & Dwyer, D.F. (2002). Degradation of trichloroethylene in wetland microcosms containing broad-leaved cattail and eastern cottonwood. Water Research., 36, 1539-46.

[6] Newman, L.A., & Reynolds, C.M. (2004). Phytodegradation of organic compounds. Current Opinion in Biotechnology., 15, 225-30.

[7] Gordon, M., Choe, N., Diffy, J., Ekuan, G., Heilman, P., Muiznieks, I., Ruszaj, M., Shurtleff, B.B., Strand, S., Wilmoth, J., & Newman, L.A. (1998). Phytoremediation of trichloroethylene with hybrid poplars. Environmental Health Perspectives., 106, 1001-4.

[8] Dudai, N., & Putievsky, E. (2006). Growth management of vetiver (Vetiveria zizanioides) under Mediterranean conditions. Journal of Environmental Management., 81, 63-71.

[9] School of Environment, Resources and Development and Faculty of Science, Kaset-sart University, (2006). Enhancement of Natural Attenuation of Soil and Groundwater Polluted by Trichloroethylene (TCE), Final Report RTG-AIT Joint Research Project, Thailand.

[10] Andra, S.S., Datta, R., Sarkar, D., Sami-nathan, S.K.M., Mullens, C.P., & Bach, S.B.H. (2009). Analysis of phytochelatin complexes in the lead tolerant vetiver grass [Vetiveria zizanioides (L.)] using liquid chromatography and mass spectrometry. Environmental Pollution., 157, 2173-83.

[11] Singh, S., Melo, J.S., Eapen, S., & D'Souza, S.F. (2008). Potential of vetiver (Vetiveria zizanoides L. Nash) for phytoremediation of phenol. Ecotoxicology and Environmental Safety., 71, 671-6.

[12] Singh, S., Eapen, S., Thorat, V., Kaushik, C.P., Raj, K., & D'Souza, S.F. (2008). Phytoremediation of 137cesium and 90 strontium from solutions and low-level nuclear waste by Vetiveria zizanoides. Ecotoxicology and Environmental Safety., 69, 306-11.

[13] Shang, T.Q., Doty, S.L., Wilson, A.M., Howald, W.N., & Gordon, M.P. (2001). Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway. Phyto-chemistry., 58, 1055-65.

[14]Schnabel, W.E., Dietz, A.C., Burken, J.G., Schnoor, J.L., & Alvarez, P.J. (1997). Uptake and transformation of trichloroethylene by edible garden plants. Elsevier Science Ltd., 31, 816-24.

[15] Ma, X., & Burken, J.G. (2003). TCE diffusion to the atmosphere in phytoremediation applications. Environ. Sci. Technol., 37, 2534-9.

[16] Chen, Y., Shen, Z., & Li, X. (2004). The use of vetiver grass (Vetiveria zizanioides) in the phytoremediation of soils contaminated with heavy metals. Applied Geochemistry., 19, 1553-65.


Sci. J. UBU, Vol. 1, No. 2 (July-December 2010) 58-64 SCIENCE JOURNAL Ubon Ratchathani University http://scjubu.sci.ubu.ac.th

Research Article

Risk Estimation of Campylobacter jejuni Caused by Chicken Meat Consumption for High Risk Group in Thailand

S. Osiriphun1, W. Koetsinchai2, K. Tuitemwong3, L.E. Erickson4, P. Tuitemwong1*

1Department of Microbiology, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand.

2Departement of Mathematics, Faculty of Science, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand.

3Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

4Department of Chemical Engineering, Faculty of Engineering, Kansas State University, Manhattan, Kansa 66506, United State of America.


Abstract

Risk estimation was developed for Campylobacter jejuni (C. jejuni) caused by chicken meat consumption for high risk group, children under 5 years of age, in Thailand. From our previous study, it was found that the more appropriate model was Beta Poisson model based on the minimum values of MSE and MAPE. The fit of the Exponential model is considered to be inadequate. In this study, Beta Poisson dose-response models are developed for describing the relationships between the numbers of C. jejuni in contaminated chicken meat that was consumed and the resulting illness outcome. Values of maximum likelihood estimated by the Beta Poisson dose response models are also determined to describe a probabilistic causal relation between exposure received and frequency as well as severity of resulting adverse health effects including number of illnesses per year. The dose-response parameters were derived from epidemiological and research data and were used to calculate the risk estimate. The number of campylobacteriosis cases caused by chicken meat consumption per year for children aged less than or equal to 5 years is predicted and risk of chicken meat consumption with C. jejuni contamination is also explored. Beta Poisson model predicted that 4,103 will become ill annually from the ingestion of C. jejuni at about 1.53 LogDose/serving. The most significant sensitivity factors that impact to incidence of C. jejuni are meal size or volume of chicken meat product per meal and the contamination levels of C. jejuni in chicken meat.

Keywords: Campylobacter, Chicken meat consumption, Children under 5 years of age, Risk Estimation.


S. Osiriphun et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 58-64


59

1. Introduction

Campylobacter is a major cause of foodborne illness worldwide especially among children. The incidence of Campylobacter enteritis in Thailand was 40,000 per 100,000 for children below 5 years of age [1]. Recent study includes work on the relative fraction of C. jejuni and C. coli isolates from Thai children during 1991 to 2000; It was found that both C. jejuni and C. coli were 75-85 percent of the total number of cases in which Campylobacter spp. was isolated from children [2]. The factor of contamination of C. jejuni in undercooked chicken meat is very large and well known that cause of campylobacteriosis illness. There are at least two ways in which carriage of C. jejuni by live poultry may affect human health: by direct infection to farm workers or processing plant operatives through handling infected poultry and through consumption of under cooked poultry meat products or by contami-nation of ready-to-eat foods [3].

2. Theory

Although, Hazard Analysis Critical Control Point (HACCP) concepts have been introdu-ced for the poultry industry in food safety control system management, Quantitative Risk Assessment (QRA) methods have become increasingly popular for the control of Campylobacter in chicken meat products. The Food and Agricultural Organization (FAO) and the World Health Organization (WHO) developed a risk assessment frame-work for Campylobacter spp. in broiler chickens [4]. Microbial risk assessment (MRA) will be the basis of food safety control systems for poultry industry in the future. About MRA in Thailand, The Agri-cultural Commodity and Food Standards of Thailand issued the first announcement of the Principles and Guidelines for the conduct of Microbiological Risk Assessment for poultry [5] which was quoted from FAO/WHO guidelines [6]. Therefore, the application of Risk Assessment to C. jejuni in processed broiler meat could lead to the prevention of

human campylobacteriosis caused by chicken meat consumption.


Epidemiological and other experimental re-sults from KMUTT Risk and Decision Analysis Lab (RADAL) were used in model development Data on Level of Contamination in Chicken Products: Cooked Chicken Meat Samples. Fifteen steamed chicken breasts and twenty chicken karaage, the restructured fried chic-ken, were collected at a processing plant. The samples were kept in an icebox and sent to the laboratory for the analysis of C. jejuni. They were analyzed using methods described by FSIS (baseline studies in poultry procedure) [7]. Dose Response for Infection: Modified Beta Poisson Model. Modified Beta Poisson model was described firstly by seven Furumoto and Mickey (1967) [8], and applied to microbial dose-response estimation by Hass (1983) [9]. From the Exponential model results; the interaction of pathogen-host can be used to describe the pathogen-host survival prob-ability by fixed value. The Beta Poisson model takes into account the variation that exists in pathogen-host interactions. If the pathogen-host survival probability is de-scribed by beta probability distribution then the probability of infection, (Pinf), can be expressed as

)12(11 /1

50inf ID

dP

(1)

where d represents the dose, α is a measure of the model’s closeness to the Poisson (Expo-nential) or pathogen infectivity (slope para-meter), and ID50 is the dose that would infect half the exposed population or the median infective dose. Given a dose response funct-ion, calculation of the ID50 is as follow [10]

Risk Estimation of Campylobacter jejuni Caused by Chicken Meat Consumption


60

)12( /150

ID

(2)

where α is shape parameter and β is scale of parameter when β≥1 and β>α.

Risk Estimation of Campylobacteriosis Ill-ness Caused by Chicken Meat Consumption. The annual risk of infection is estimated by Eq. (2) [11]

nIAI dPdP )(11)()(

(3)

where PI(d) is the risk of infection in an individual after ingestion of a single pathogen dose d; PI(A)(d) is the annual risk of infection in an individual from n exposures to the single pathogen dose d per year.

Statistical Method for Sensitivity Analysis of Risk Estimation. Regression analysis is the statistical method for sensitivity analysis and can be employed as a probabilistic sensitivity analysis technique. The regression model application is available in @Risk software package (Palisade, NY) [12].

In our study, statistical method for sensitivity analysis was used, the method involves running simulations in which inputs are assigned probability distributions and assess-ing the effect of variance in inputs on the output distribution [11].


Concentration of C. jejuni in Chicken Meat Products. C. jejuni was not found in any steamed chicken breast and chicken karaage samples collected from the processing plant. As a result, information and data of the dose response model development were obtained from experimental data reported by Iamta-weejaleon (2009) [14], Kooprasertying (2009) [15], and Tuitemwong (2009) in our laboratory (Please see in Osiriphun, 2009 [16]). The estimate of the Beta-Poisson dose

response curve for Thai high risk group was carried out in our previous study [16]. ID50 or ND50 and alpha values for Beta Poisson model were calculated. The parameter values used to predict the dose response curve are presented in Table 1.

Table 1. Summary of parameters used in Beta Poisson dose response model.

Data set

Beta Poisson Source of parameter

Study

Data Reference

1 alpha= 0.2298

ID50=49,377

Calculated Padungtod, 2003

2 alpha= 0.2219

ID50=32,554

Calculated Thai Epidemiologica

l data

*ID50 is the median infective dose

In Table 1, alpha values of data set 1 and 2 for Beta Poisson model are 0.2298 and 0.2219, respectively; ID50 value for data set 1 is 49,377, ID50 for data set 2 is 32,554, respectively.

Risk Estimation of High Risk Population (Children under Five Years of Age). The dose-response parameters were used to calculate the risk estimate. Table 2 summar-izes the predicted daily dose estimation of C. jejuni and the predicted daily risk of illness. From the dose response relationships, the imputed daily dose may be computed given the daily and annual risk. It was found that, from the best estimates of the dose-response curve, the individual risk resulting from chicken meat consumption 33.86 g/day of the product with C. jejuni levels of 0-5.53 LogCFU/g would be illed from zero to 5,284 persons per year. PI(A) is the annual risk of infection in an individual from n exposures to a single pathogen dose d per year. Assume that all infections will lead to campylobact-eriosis disease. Results from the Beta Poisson model associated with two sets of data are illustrated in Figure 1.

S. Osiriphun et al., Sci. J. UBU, Vol. 1, No. 2 (July-December 2010) 58-64


61

Table 2. Probability of illness per serving for the high risk population estimated for different levels of C. jejuni at the time of consumption and the estimated number of cases per year in Thailand.

Beta Poisson model

Actual model

C. jejuni

(CFU/g)

Maximum Dose

(CFU) (1)

LogDose

(LogCFU/Serving)

Risk of

illness/year

(PI*PE) (2)

Estimated

annual

number of

cases

Risk of

illness/year

(PI*PE)

Estimated

annual

number of

cases

0.00E+00 0.00 0.00 0.000 0 0.000 0

1.00E+00 33.86 1.53 0.197 5,284 0.153 4103

1.00E+01 338.60 2.53 0.097 1,097 0.097 1097

1.00E+02 3386.00 3.53 0.042 210 0.042 210

1.00E+03 33860.00 4.53 0.019 42 0.019 42

1.00E+04 338600.00 5.53 0.009 9 0.009 9

NOTES: (1) Serving size of 33.86 g. (2) Using the risk from a dose of 1 CFU as reference.

0

5,284

1,097

21042 90

4,103

1,097

21042 9

0.E+00

1.E+03

2.E+03

3.E+03

4.E+03

5.E+03

6.E+03

0.00 1.53 2.53 3.53 4.53 5.53

Campylobacter jejuni (LogDose per gram per serving)

Num

ber

of C

ampy

loba

cter

isos

is c

ases

(H

igh

risk

pop

ulat

ion)

B-P Model B-P Actual

Figure 1. Number of estimated campylobacteriosis cases per year from Beta Poisson model and actual data. For data sets 1 and 2 associated with children under 5 years old in Thailand. The Minimum Level of Error (MLE) values from the Beta Poisson dose response models were used to describe the probabilistic causal relation between exposures received. The comparison of campylobacteriosis cases obtained from the two sets of data are shown in Figure 1, the Beta Poisson model associated with data set 1 suggests that there were 5,284, 1,097,

210, 42, and 9 consumers become ill from the ingestion of C. jejuni at 1.53, 2.53, 3.53, 4.53 and, 5.53 LogDose/serving, respectively. The Beta Poisson model with data set 2 gave values of 4,103, 1,097, 210, 42, and 9 for annual illness rate from the consumption of C. jejuni at 1.53, 2.53, 3.53, and 4.53 logDose/serving, respectively. The results of the model are also comparable with epidemiological evidence. The predicted



62

number of C. jejuni from Beta Poisson and actual model conformed to that of the outbreak of C. jejuni caused by chicken casserole dish at a restaurant in Australia. Numbers of C. jejuni in the chicken dish ranged from 53 to 750 CFU per cm2 (1.72-2.88 LogDose per cm2) [17]. Predicted number of C. jejuni causing illness for at least one person was 2.55 logDose/serving; it was not different from the outbreak data. The outbreak data and predictive model presented the relative low infective dose of C. jejuni.

Risk Estimation

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

Campylobacter jejuni (Log CFU per serving size)

Ris

k of

illn

ess

Beta-Poisson (model data) Padungtod (2003) (Actual data) Graphical comparison (Figure 2) showed that the highest risk approximation from consum-ing chicken meat contaminated with C. jejuni from Beta Poisson dose response model and the actual data from [18] were in the same line at 1.65 Log Dose per serving. The highest risks of illness caused by chicken meat consumption were 0.198 and 0.156 for model data and actual data, respectively. The results were not over or under estimated when compared with the two studies on outbreak data for C. jejuni associated with chicken meat consumption. In Japan, the outbreak data of gastroenteritis occurred among 3rd year students of a high school in Chiba Prefecture, Japan, after they consumed the cooked chicken for their lunch. The percentage of illness by C. jejuni among the students in classes A, B, and E were 2.78 %, 11.43 %, 11.90 %, respectively [19]. In an outbreak of Campylobacter enteritis at a restaurant in Australia, a chicken casserole dish was implicated with a food-specific

attack rate of 58%. C. jejuni was isolated from 3 of 4 symptomatic patients and from three raw fresh chicken samples closely associated with the implicated chicken. Concentrations of C. jejuni in the chicken casserole dish ranged from 53 to 750 CFU or 1.72-2.88 LogDose per square centimeter of surface area (5,300-75,000 CFU or 3.72-4.88 LogDose per serving) [17]. Another outbreak report is C. jejuni contamination in stir-fried unmarinated chicken pieces occurred in Cardiff, Wales in 1997. The authors suggested that the significant dose-response relationship between risk of illness and amount of chicken consumed was caused from undercooking of chicken combined with inadequate cooking time due to the use of large chicken pieces [20].

Statistical Sensitivity Analysis of Risk Estimation. The regression sensitivity for cases per year was used in order to rank sensitivity. The spearman ranking coefficients for meal size, contamination level of C. jejuni in chicken meat product, meals per million consumers per year, ingested dose and ID50 were 0.801, 0.575, 0.009, -0.016, and -0.077, respect-ively. The most significant sensitivity factors on the impact to incidence of C. jejuni for children below 5 years of age in Thailand were meal size or volume of chicken meat product per meal and the contamination levels of C. jejuni in the chicken meat. It means that a lower meal size results in a lower incidence of campylobacteriosis.

0.009

-0.016

-0.081

0.575

0.801

-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

Std b Coefficients

MealSize/C9

Contamination/C6

ID50= / (Predicted)/E7

Dose / RiskPoisson(3962)/J4

Meals per million per year.../C24

Figure 2. Risk estimates for data sets 1 and 2 from Beta Poisson dose response model.

Figure 3. Regression sensitivity analyses for cases per year.

S. Osiriphun et al., Sci. J. UBU, Vol. 1, No. 2 (July-December 2010) 58-64


63

The factor of C. jejuni contamination in undercooked chicken meat containing C. jejuni is very large and well known that cause of campylobacteriosis illness. There are at least two ways in which carriage of C. jejuni by live poultry may affect human health: by direct infection to farm workers or processing plant operated through handling infected poultry and through consumption of under cooked poultry meat products or by cross contamination of ready-to-eat foods [3]. 5. Conclusions

The estimated risk of campylobacteriosis dueto the consumption of chicken meat products is 0.198 with level of C. jejuni at 1.65 LogDose per serving. The most significant sensitivity factors that have the impact to incidence of C. jejuni for children below 5 years of age in Thailand are meal size or volume of chicken meat product per meal and the contamination levels of C. jejuni in chicken meat. The recommendation for food service operators and home kitchen,

the hygiene measures should be aimed at minimizing cross-contamination between raw chicken and hands, contact surfaces and utensils [19]. The suggested interventions were to prevent cross contamination and to cook the smaller size of raw chicken with the minimum internal temperature to 74°C to provide the acceptable risk [21].

Acknowledgements

We would like to thank Mrs. Leelaowadee Sangsuk, medical scientist from Anaerobic Section, National Institute of Health, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand and Mrs. Ladaporn Bodhidatta, scientist from Department of Enteric Disease, Armed Force Research Institute of Medical Sciences, Bangkok, Thailand, for valuable data and information which used to evaluate the model of this study. The research was funded by the National Research Council and Faculty of Science, KMUTT, Thailand.

References

[1] Taylor, D.E., (1992). Campylobacter infect-ions in developing countries. In I. Nachamkin, M.J. Blaser, & L.S. Tompkins (Eds), Campylobacter jejuni: Current Status and Future Trends (pp. 9-19). Washington, USA: American Society for Microbiology.

[2] Serichantalergs, O., Dalsgaard, A., Bodhidat-ta, L., Krasaesub, S., Pitarangsi, C., Srijan, A., & Mason, C.J. (2007). Emerging fluoro-quinolone and macrolide resistance of Cam-pylobacter jejuni and Campylobacter coli isolates and their serotypes in Thai children from 1991 to 2000. Epidemiology and Infection, Vol.135, (8), 1299-306.

[3] Saleha,A.A., Mead, G.C., and Ibrahim, A.L. (1998). Campylobacter jejuni in poultry production and processing in relation to public health. World’s Poultry Science Journal, Vol. 54, 49-58.

[4] FAO/WHO (2001), Preliminary report of hazard identification: Hazard characterizat-ion, and exposure assessment of Campylo-bacter spp. in broiler chickens.

[5] ACFS (2007). National bureau of agricult-ural commodity and food standards: Principles and guidelines for the conduct of microbiological risk assessment [PDF]

[6] FAO/WHO (2001). Principles and guidelines for the conduct of microbiological risk asse-ssment (CAC/GL-30). In Joint FAO/-WHO food standards programme, codex aliment-arius commission: food hygiene basic texts, (2nd , pp. 55-64). Rome, Italy: FAO.

[7] NACMCF, National Advisory Committee on Microbiological Criteria for Foods (2007). Analytical Utility of Campylobacter Metho-dologies. Journal of Food Protection, 70(1), 241–50.

[8] Furumoto, W.A. & Mickey. R.A. (1967). A mathematical model for the infectivity-dilution curve of tobacco mosaic virus: theoretical considerations, Virology, 32, 216-23.

[9] Haas, C.N. (1983). Estimation of risk due to low doses of microorganisms: a comparison



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of alternative methodologies. American Jour-nal of Epidemiology, 118, 573-82.

[10] Haas, C.N, 2003, Dose-response modeling for microbial risk In R.H., Schmidt, & G.E. Rodrick (Eds.), Food Safety Handbook (pp.47-57). New Jersey, USA: John Wiley & Sons, Inc.

[11] Mara, D.D., Sleigh, P.A., Blumenthal, U.J., & Carr, R.M. (2007). Health risks in wastewater irrigation: Comparing estimates from quantit-ative microbial risk analyses and epidemiolo-gical studies. Journal of water and Health, 39-50.

[12] Palisade, Inc., (2003). Guide to using risk: Risk analysis and simulation add in for microsoft excel. New York, USA: Newfield.

[13] Frey, C., Mokhtari, A., Danish, T. (2003). Evaluation of selected sensitivity analysis: Methods based upon applications to two Food safety process risk models. USA: Department of Agriculture.

[14] Iamtaweejaloen, P. Tuitemwong, P., Kun-kreangvong, J., & Tuitemwong, K. (2009). Reduction of Campylobacter jejuni in chick-en carcass using chlorine and ozone. In the 47th Kasetsart University Annual Confer-ence: Agro-Industry, 17-20 March 2009, pp. 655-62.

[15] Tuitemwong, P., Kunkreangvong, J., & Tui-temwong, K. (2009). Reduction of Listeria monocytogenes in poultry process using

ozonation system. In the 47th Kasetsart University Annual Conference: Agro-Industry. 17-20 March 2009, pp. 647-54.

[16] Osiriphun, S., Koetsinchai, W., Tuitemwong, K., Erickson, L.E., & Tuitemwong, P., 2009, Campylobacter jejuni Incidence, infection, and illness in Thailand, Journal of Food Safety.

[17] Rosenfield, J.A., Arnold, G.J., Davey, G.R., Archer, R.S., & Woods, W.H. (1985). Serotyping of Campylobacter jejuni from an outbreak of enteritis implicating chicken. Journal of Infection. 11, 159– 65.

[18] Padungtod, P., 2003. The comparative of Salmonella and Campylobacter in Northern of Thailand. Faculty of Veterinary Medicine, Chiang Mai University.

[19] Yoda, K. and Uchimura, M. (2006). An outbreak of Campylobacter jejuni food poisoning caused by secondary contamination in cooking practice at a high school. Japanese Journal of Infectious Disease, Vol. 59, 408-9.

[20] Evans, M.R., Lane, W, Frost, J.A., & Nylen, G. (1998). A Campylobacter outbreak associ-ated with stir-fried food. Epidemiology and Infection, 121(2), 275-9.

[21] FAO/WHO (2009). Microbiological risk ass-essment series 19, Salmonella and Campy-lobacter in chicken meat (Meeting Report) [PDF].




Research Article

Preparation of Purified Dye Powder from the Bark of Livistona speciosa

P. Muangthai*, N. Promrong, C. Wannawong

Department of Chemistry, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand.


1. Introduction

There are many dyes which were prepared from natural source as plants and animals [1]. Many parts of the plants such as stem, leaf, bark, root, seed, and flower were used as raw materials for dye extraction [2]. All those dyes were freshly prepared for dyeing on fibers by boiling the part of plant for

approximately 1hour. There is a few work that prepare the dye as a solid powder form [3]. The natural dye pigment from plant trends to decrease for using in the dyeing industries since its stability on dyeing onto fiber [4] and a small quantity of their extraction yields [5]. The aim of this work was to prepare the natural dye pigment as a powder from the bark of the Livistona speciosa by using 4 solvents in extraction the pigment such as water, methanol, 3% acetic acid, and 0.5 M

Abstract

Khor is the plant in Palm family. Its scientific name is Livistona speciosa. The local people from the Northern East of Thailand use it as the raw material to prepare dye. In this work the dye was extracted from the bark of Livistona speciosa by using 4 solvents in extraction such as water, methanol, 3% acetic acid and 0.5 M sodium hydroxide solution. Then those dyes were purified by adsorption on 3 adsorbents such as a marly limestone, silica gel and bentonite clay. Finally, the dye on each adsorbent was removed by dissolved in water, filtered through to filter paper out, and evaporated to dry dye powder. The efficiencies of adsorption on cotton fiber of those dyes were also studied. The results showed that the extracted dye solution from 4 solvents had different colors, and the dye could be purified by 3 adsorbents. The methanol solution was the best solvent for extracted dye which was purified by bentonite clay, gaven the highest percentage yield of purified dye as pale green brown color powder approximately about 46.87%. The efficiency of dye adsorption on cotton fiber which was extracted by methanol and purified by silica showed the highest %E at 88.76 %.

Keywords: Adsorbent, Dye powder, Livistona speciosa.



66

of sodium hydroxide solution. Then the extracted crude dyes were purified by adsorbents: marlylime stone, silica gel and bentonite clay. Then the efficiency of adsorption on cotton yarn fibers were also studied. 2. Theory

Livistona speciosa is one type of plant in palm family which was called in many common names as Khor. This plant was used as a raw material to prepare an orange brown color dye solution for dyeing cotton fiber of Kalasin Province [6]. There are many reports about the dye from Thai wisdom plant such as Clitoria ternatea L. or common name as unchan, marigold [7,8] etc. This work was planned to prepare the dye in powder form from Livistona speciosa., as a variety color since the local people still use the fresh plant to prepare the dye solution in dyeing process which was inconvenience in practice. The bark of Livistona speciosa was extracted the crude dye solution by using 4 solutions such as hot water, organic alcohol solvent (methanol), acetic acid solution and sodium hydroxide solution. Then, those dye solutions were purified by comparing the efficiencies of 3 adsorbents such as marly limestone, silica gel and bentonite clay since the dye solutions from many plants contain many components as lignin, pectin etc. [9]. Other components effect on the preparation of pure dye powder. Thus in the work, the above three adsorbents were selected to purify the crude dye. In generally marly limestone contains calcium carbonate as a main component [10] which was used for adsorption of some ions or molecules in substance or use as pH treatment [11]. Silica gel is an important adsorbent for a chromato-graphic technique but it expensive cost. Bentonite clay is a dark-grey to dark-green clay-rich rock which is composed of mostly montmorillonite, with a minor conponent of cristobalite, zeolite, and quartz, among others. Bentonite formation occurs when volcanic ash is altered by low-grade hydro-thermal alteration [12]. The structure of bentonite is an aluminium phyllosilicate as,

(Na,Ca)0.33(Al,Mg)2Si4O10(OH)2·nH2O. Ben-tonite was used in many industries as food and cosmetics industry [13]. All above 3 types of adsorbents may differ in their adsorption efficiencies. Finally, the dye was removed out and prepared as dye powder. The powder of those dyes were used to study the efficiency of adsorption on cotton fiber [14]. The dye powder from Livistona speciosa which was extracted and adsorbed on difference medium should gave the purified dye powder in difference yield. The percentage of the dye powder was calculated from Eq. (1)

g. of purified dye% dye powder = 100

g. of Livistona speciosa

(1) The intensity of purified dye powder could be also monitored by measuring the absorbance using visible spectrometry technique [15]. The quality of dye powder could be tested by evaluate the % exhaustion (%E): fixing the dye on cotton fiber and calculating the efficiency of fixing or the exhaustion from Eq. (2).

0 1

0

% 100Abs Abs

EAbs

(2)

where 0

Abs is an absorbance of dye

solution at initial before dyeing process, and

1

Abs is an absorbance of dye solution after

dyeing process.


Part1: Extraction of Dye from the Bark of Livistona speciosa. The branch of Livistona speciosa (from Bann Thung Phoo, Kalasin Province, Thailand) was crushed and chopped into small pieces. Then 100 g of Livistona speciosa was weighed, and filled with 300 ml of pure water and heated to boil for 30 min. The residue plant was filtered out to collect the dye solution. The crude dye was heated again to remove the water, and weighed. The other portion of Livistona speciosa was treated as above but extracted

P. Muangthai et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 65-70


67

for the dye solution by using methanol (laboratory grade purchased from Carlo Erbra), 3% acetic acid (laboratory grade purchased from BDH) and 0.5 M of sodium hydroxide solution (laboratory grade purchased from Merk). Part2: Preparation of Dye Powder. The 25 ml. of extracted dye solution from part 1 was poured into the 20 g of dried marly limestone (laboratory grade purchased from BDH). The dye adsorbed on the adsorbent, and dried by hot air oven (Buchii) at 80 oC for 1 hour. The adsorbed dye on each adsorbent was dis-solved with distilled water, the residue adsorbents were filtered out. The purified dye solution was evaporated to dryness, and weighed the dried powder for calculate the percentage yield of dye by Eq. (1). Part3: Analysis the Exhaustion, (%E) of Dye Powder. The white color cotton fiber (from Bann ThungPhoo, Kalasin Province, Thai-land) was placed into 1% of sodium hydroxide solution to purify the fiber before dyeing process. The 4 g of purified white cotton fiber was dipped into a purified dye solution, and boiled with stirring for 1 hour [16]. The color cotton fiber was removed from the residue solution. The residual color solution was measured for an absorbance maximum at maximum wavelength by Ultraviolet visible spectrophotometer (UV-2450 PC double beam spectrophotometer) compare with the color of dye solution before dyeing process. The efficiency of fixing or the exhaustion was calculated from the following Eq. (2).


From part 1, the dye solution which was extracted from Livistona speciosa, by using 4 solvents, gave the difference color solution as shown in the Table 1. The color of extracted dye presented in the orange brown color to a dark brown color. However, the color from Livistona speciosa bark also depends on the freshness of the bark. The color line in hot

tone color even it was extracted by different solvent. The methanol solvent was given the best extraction from the plant but may extract many organic compounds from Livistona speciosa bark too.

Table 1. Colors of extracted dye solution.

Solvent Color of Extracted Dye Solution

Hot Water Pure Methanol 3 % Acetic Acid0.5 M Sodium Hydroxide

Orange Brown Orange Brown Dark Brown Red Dark Brown

After the extracted dye solution was prepared to powder by purification on the 4 types of adsorbents, the adsorbent adsorbed only the dye on their surface which showed the different color as present in Table 2. Table 2. Colors of dye that adsorbed on adsorbents.

Color on Adsorbent Solvent Marly Lime- stone

Silica Gel

Bentonite Clay

Hot Water Grey Brown Pale Brown

Pure Methanol

Pale Pink

Brown Pale Brown

3 % Acetic Acid

Pink Brown Pale Brown

0.5 M Sodium Hydroxide

Pale Brown

Red Brown

Brown Black

The color of marly limestone which adsorbed the crude dye from each solvent showed a pale pink color to a brown color. This showed that the calcium carbonate which was the major component of marly limestone trapped the dye at acid site of dye on its alkali surface. Silica gel (the general adsorbent) used for thin layer chromatographic showed a brown color to a red brown color; however, silica gel structure contained 3 silanol groups. The silica gel surface could adsorb many substances on the surface by adsorption at those silanol sites. Bentonite clay commonly



68

has grey color powder in natural, after the adsorption process it showed pale brown color to dark brown from extracted dye. Thus, the results from Table 2 presented that all adsorbents could prepare the dye powder as many colors, this means that variety colors of dyes could be prepared from Livistona speciosa bark.

Table 3. Colors of purified dye powder.

Color of Purified Dye Powder Solvent Marly Lime-stone

Silica Gel

Bentonite Clay

Hot Water Brown Brown Dark Brown

Pure Methanol

Brown Brown Pale Green Brown

3 % of CH3COOH

Brown Orange Green Brown

0.5 M of NaOH

Dark Brown

Dark Brown

Dark Brown

Table 4. The percentage yields of purified dye powder.

% Yield of Purified Dye Powder

Solvent

Marly Lime- stone

Silica Gel

Bentonite Clay

Hot Water 31.75 32.96 32.14

Pure Methanol

34.93 40.45 46.87

3 % of CH3COOH

41.34 38.56 37.99

0.5 M of NaOH

42.03 43.05 41.81

The color from the adsorbed adsorbents was removed out from their surface by dissolve in pure water again. However, from this part of the experiment, the other impurity was still adsorbed on the surface of adsorbent, and only purified dye was dissolved again with pure water, and prepared as solid purified dye as shown in Table 3. From the table, the

purified dye powder from Livistona speciosa showed brown to dark green brown colors. The percentage yields of purified dye powder were presented as in Table 4. The percentage yields of those dye powder in the table showed that 0.5 M sodium hydroxide solution which purified by 3 adsorbent was given the highest yield at 41.81–43.05%. This showed that sodium hydroxide which was an alkaline medium was the best solvent to be an extracted dye solution from Livistona speciosa. In addition, silica gel was the best absorbent to be a purified dye solution since it showed the highest percent-age yield at 43.05%. The base condition was an appropriate condition to extract dyestuff from this plant. However, pure methanol extraction gave the highest percentage yield at 46.87%.Since the color in all plant is an organic compound [17] especially, Livistona speciosa may be an anthocyanin and its derivatives since the absorption spectrum showed the absorbance maximum at 480-481 nm as shown in Figure 1. In the figure, the spectrum of dye showed broad band spectrum, so it means that the crude dye contained other components.

The results from the study of the efficiency of dye in fixing on cotton dye fiber by measuring the exhaustion, (%E) of the dye powder values as presented in Figure 2.

Figure 1. The absorption spectrum of dye powder from Livistona speciosa.

P. Muangthai et al., Sci. J. UBU, Vol. 1, No. 2 (July-December, 2010) 65-70


69

The %E of dye powder which extracted by methanol which purified by silica gel was given the highest % exhaustion at 88.76, this means that the dye powder from silica gel has the most effective on dyeing process. In chromatography technique, the silica gel was also used as a standard adsorbent [16], but it was expensive. The bentonite clay adsorbent was also presented the high value of %E in the range of 85.75% by water extraction, so this is a good result to prepare the dye powder on bentonite which it is a cheaper adsorbent than silica gel. However, the dyeing process in this experiment use the direct dyeing with no using of any mordant, but in the real system the mordant must be added in the dyeing process for better fixing dye on fiber. This work showed that the dye from the Livistona speciosa could prepared as dye powder which was purified by the 3 adsorb-ents. The best condition to extract dye should

use a methanol solution as extracted solvent since this condition was given the highest percent-age yield of dye powder. The minor case may extract the crude dye from the bark of Livistona speciosa by hot water solvent and be purified by bentonite clay which was also gave %E at 85%.

5. Conclusions

The dye powder from the bark of Livistona speciosa or Khor should be used to prepare the crude dye by using 4 solvents such as water, pure methanol, 3% acetic acid solution and 0.5 M. sodium hydroxide solution which was given the orange brown to red dark brown color solutions. After the crude extraction of dye solutions, the dye were purified by using 3 adsorbents such as marly limestone, silica gel and bentonite clay. Then they were redissolved to distill water, and evaporated to dryness, given the drak brown to brown color of dry powder. The percentage yield of dye powder was 36.75–46.87%, given the strong absorption peak at 480-481 nm. The dye powder showed the % exhaustion with no mordent at 88.76% by using methanol as extracted solvent which was purified by silica gel. However, the extraction of crude dye from the bark of Livistona speciosa by hot water, which was purified by bentonite, was also given the high %E about 86 %.

Acknowledgements

The authors grateful to Srinakharinwirot University for the financial support on 2009 for this research.

References

[1] Bulda, O.V., Rassadina, V.V., Aleksei chuk, H.N., & Laman, N.A. (2008). Spectrophoto- metric measurement of carotenes, Xanthoph-

ylls, and chlorophylls in extracts from plant seeds. Russian Journal of Plant Physiology, 55(4), 544-51.

Figure 2. The exhaustion, (%E) of purified dye powder.



70

[2] Kamel, M.M., Youssef, B.M., & Magda, M.K. (1991). Adsorption of anionic dyes by kaolinites. Dyes and Pigments, 15(3), 175-82. [3] Thomas, B., Amalid, M.A., & Rita , M.

(2007). Natural dyes for textile dyeing: A comparison of methods to assess the quality of Canadian golden rod plant material. Dyes and Pigments. 75(2), 287-93.

[4] Ranjana, B., & Saikia, C.N. (2005). Isolation of colour components from native dye bearing plants in northeastern India. Bioresource

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Balachandar, V, Lakshmanaperummalsamy, P., Chan Chae, J., & Taek, O.B. (2009).

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[7] Chuakul, W., Saralamp, P., & Temsirinikkul (1997). Thai Medicinal Plants,Vol 1. Bang-kok: Bmarin Printing Printing and Publishing Co.,Ltd.

[8] Epolito, W.J., Lee, Y.H., Bottomley, L.A., & Pavlostathis, S.G. (2001). Characterization of the textile anthraquinone dye Reactive Blue 4. Dyes and Pigments, 75(2),157-63. [9] Silke., K., Kornmüller, A., & Jekel, M. (2001). Screening of commercial sorbents for the removal of reactive dyes. Dyes and

Pigments 51(2-3), 287-93. [10] Dazhong, S., Fan, J., Zhou, W., Baoyu , G., Qinyan,Y., & Kang, Q. (2009). Adsorption kinetics and isotherm of anionic dyes onto organo-bentonite from single and multisolute systems . Journal of Hazardous Materials, 172(1), 97-107. [11] Thorp, J.M, & Wool, J.B. (1967). Modific- ation of the porous structure of silica gel on purification and the associated change in the dielectric behaviour of adsorbed benzene. Trans. Faraday Soc., 63, 2068-78. [12] March, J. (1992). Advanced Organic Chem- istry (5th eds.). New York: Wiley & Sons. [13] Schurrenberger, D., Russell, J., & Kerry K. (2003). Classification of lacustrine sediments based on sedimentary components. Journal of Paleolimnology, 29, 141- 54. [14] Derek,. J.C. (1981). Exclusion chromatography of anionic dyes : Anomalous elution peaks due to reversible aggregation. Jour- nal of Chromatography A, 209(3), 170-86. [15] Parkes C.H. (2003). Creating color in Yarn: An introduction to natural Dyes, Knitters Review. [16] Klein, P.D (1961). Silica gel structure and the chromatographic process: Effect of pore diameter on the adsorption and differential migration of sterol acetates. Analytical Chemistry, 33 (12), 1737-41. [17] Das, S. (1992). Application of natural dyes on silk. Colourage, 19(9), 52-4.


Sci. J. UBU, Vo1. 1, No. 2 (July-December, 2010) 71-77 SCIENCE JOURNAL Ubonratchathani University http://scjubu.sci.ubu.ac.th


Research Article

Continuous Cheese Whey Fermentation in a Series of Two Reactors

R. Agustriyanto*, A. Fatmawati

Chemical Engineering Department, Surabaya University, Surabaya 60292, Indonesia.


1. Introduction

In recent years, it is recognized that the global crude oil reserve is finite and its depletion is occurring much faster than before. The shortage of fossil fuel has encouraged the investigation on some alter-native energy sources. Bio-ethanol is one of the alternative energy sources, which can be produced from fermentation. Currently, the global ethanol supply is produced mainly from crop products such as corn, sweet sorghum, and sugar cane. Hence, to produce ethanol, it always needs land

opening for plantation which ultimately will result in deforestation. To reduce land utilization for plantation, and to eliminate the land competition between food and energy orientation, the use of alternative non-crop raw materials needs to be explored. Such raw materials could be from industrial waste.

Many researches have been concerning the use of cheese whey fermentation to produce ethanol [1-5]. The effect of operating parameters such as initial pH, cheese whey powder (CWP) concentration, and external nutrient (N,P) supplementation on the cheese whey powder (CWP) fermentation has been investigated by Kargi and Ozmihci

Abstract

A steady state model for cheese whey fermentation in two series of reactors has been developed. The model was derived based on mathematical and fundamental chemical engineering concept. The aim of this research is to develop the model for cheese whey fermentation in two series of reactors and to investigate the effect of hydraulic retention time on process performance. This research showed that the recommended values of hydraulic retention time for the second reactor decreased as the inlet substrate concentration increased. The results were 14, 7, and 2 h for 50, 100, and 150 g/l inlet substrate concentration. Another important result was the significant reduction of substrate and slightly higher values of ethanol concentration obtained by combining two reactors in series compared to the use of single reactor.

Keywords: Bio-ethanol, Cheese whey, Fermentation, Reactor.



72

[3]. They used cheese whey powder as the substrate of batch fermentation and found that initial pH of 5 was the most suitable for producing maximum final ethanol concent-ration and ethanol formation rate. The external addition of N and P source did not improve the ethanol formation. The final ethanol concentration and ethanol formation rate increased with sugar concentration. The ethanol production from batch fermentation of crude whey by Kluyveromyces marxianus has been investigated by Zafar and Owais [5]. They reported that the specific cellular growth rate and product formation rate reached maximum values of 0.157 and 0.046 1/h at exponential phase. Ghaly and Taweel have studied the kinetic of batch and continuous fermentation of cheese whey by yeast Candida pseudotropicalis [1-2]. They produced kinetic parameter from batch fermentation and recommended the operat-ing parameters for continuous cheese whey fermentation that gave the maximum ethanol concentration are 150 g/L inlet substrate concentration and 42 h hydraulic retention time. Agustriyanto & Fatmawati [4] found that low value of hydraulic retention time is favoured in single fermenter. Ethanol productions from sweet whey permeate and sweet whey permeate-grain batch fermentations have also been investigated [6]. The yeast cells used were Kluyveromyces fragilis and Saccharomyces lactis. The ethanol concentration produced from 24 h whey permeate fermentation was 20 g/L. As much as 97 and 94 g/L of ethanol was produced from whey permeate-grain fermentation using yeast K. fragilis and S. cerevisiae respectively in 36 h.

2. Theory

Figure 1 shows the two series reactors system which is used in the modeling. Cheese whey substrate inlet flow with initial lactose concentration Si enters the first fermenter at constant volumetric flow rate of Q. Ethanol will appear in first and the second reactor product stream with concent-ration P1 and Po respectively as a result of

lactose fermentation by the cell (Candida pseudotropicalis). The Concentrations of the cell in the inlet, first and second reactor outlet are denoted as Xi, X1, and Xo respectively and the remaining lactose at the first and second reactor outlet are S1 and So. At certain inlet volumetric flow rate, the ethanol product concentration will depend on the volume of the two reactors.

Figure. 1 Two series reactors for cheese whey fermentation.

2.1. Mathematical Modeling of the First Reactor

A. Growth Kinetic. Optimal expression of growth kinetics depends on the transport of the necessary nutrients to the cell surface, the rate of mass transfer from the medium into the cells and the environmental para-meters (temperature and pH) being opti-mally maintained. The kinetic of microbial cell growth can be modeled mathematically as follows [7-8]. X

R X (1)

where is the specific growth rate (1/h)

and X is the cell concentration (g/L).

At high substrate concentration, cell growth rate could be inhibited by the substrate. Fermentation product can also cause cell growth inhibition. Ethanol as a fermentation product is well known to be inhibitory to both yeast cell growth and ethanol prod-uction. The effect of inhibition must be

R. Agustriyanto et.al., Sci. J. UBU, Vo1. 1, No. 2 (July-December, 2010) 71-77


73

accounted in the growth model. One of the models of specific growth rate which involve the effect of substrate and product inhibition is shown below

pm s

s s p

KS K

K S K S K P

(2)

wherem

is the maximum specific growth

rate (1/h), S is the substrate concentration (g/L), P is the ethanol concentration (g/L),

sK is the saturation constant (g/L), '

sK is

the substrate growth inhibition concentrat-

ion (g/L), and P

K is the ethanol growth

inhibition concentration (g/L).

B. Cell Mass Balance. The cell mass balance in continuous fermenter can be formulated as follows

[Cell accumulation rate] = [cell input rate] + [cell growth rate] – [cell death rate] – [cell output rate] (3) Mathematically, the above mass balance can be rewritten below

i d

dXV QX XV K XV QX

dt (4)

or

1( )

i

pm s

d

s s p

dXX X

dt R

KS KX K X

K S K S K P

(5)

where d

K is the specific cell death rate,

(1/h), V is the fermenter volume (L), Q is

the volumetric feed rate (L/h) and R is the hydraulic retention time (h) which can be written as

V

RQ

(6)

Assuming that the feed is sterile ( 0i

X )

and the fermenter is at the steady-state condition, the above equation can be written as

1 pm s

d

s s p

KS KK

R K S K S K P

(7)

C. Substrate Mass Balance. Using similar formula of cell mass balance, the mass balance of substrate in continuous ferment-ation can be written as follows

[Substrate accumulation rate] = [input rate of substrate] – [substrate utilization rate for growth] – [substrate utilization rate for maintenance] – [substrate utilization rate for product] – [output rate of substrate] (8)

1

i

SX Sm SP

dSV QS

dt

R R R V QS

(9)

where i

S is the substrate concentration at

the inlet concentration of the first reactor,

(g/L), 1

S is the substrate concentration at the

outlet concentration of the first reactor

(g/L), SX

R is the substrate utilization rate for

cell growth (g/L.h), Sm

R is the substrate

utilization rate for maintenance (g/L.h) and

SPR is the substrate utilization rate for

product format-ion (g/L.h).

At steady-state condition, we can modify Eq. (9) into the following expression



74

1

/ /

( ) X

i S

X S P S

R XXS S R m X

Y Y

(10)

where /X S

Y is the yield coefficient for cell

on substrate, S

m is the maintenance energy

coefficient (1/h), is the growth associated product formation constant which can be written as

/

/

P S

X S

Y

Y (11)

is the non-growth associated product

formation constant (1/h), which can be neglected because ethanol is a growth asso-ciated product. Hence, we can simplify (10) into the following

1

/ /

( ) X

i S

X S P S

RXS S R m X

Y Y

(12)

D. Product Mass Balance. Finally, the mass balance for ethanol as the product of the fermentation can be derived

[Ethanol accumulation rate] = [input rate of ethanol] + [ethanol production rate] – [ethanol output rate] (13)

or

1i X

dPV QP R X V QP

dt (14)

At steady state and no product in the input flow the above equation can be simplified as follows:

pm s

s s p

KS KP RX

K S K S K P

(15)

2.2. Mathematical Modeling of Second Reactor

Using the same method as for the first reactor, the following models can be obtained.

* Cell Mass Balance:

1

1

XX

XKPK

K

SK

K

SK

S

R

dp

p

s

s

s

m

(16)

* Product Mass Balance:

1

pm s

s s p

KS KP P RX

K S K S K P

(17)

These two balance equations are slightly different from the first reactor due to the presence of cell and product in the inlet flow of the second reactor; while the specific growth rate equation and substrate balance are remain the same.

Table 1. Kinetic parameters.

Initial Substrate Concentration

50 g/L 100 g/L 150 g/L

m 0.0510 0.0510 0.0510

sK 1.9000 1.9000 1.9000

pK 20.6500 20.6500 20.6500

/X SY 0.0480 0.0480 0.0380

/P SY 0.4260 0.4420 0.4240

dK 0.0022 0,0032 0.0041

Sm 4.2100 4,0400 4.1800

sK 112.5100 112.5100 112.5100

The kinetic data for the fermentation of cheese whey by Candida pseudotropicalis has been previously published [1] and is shown in Table 1.



75


Micoorganism and Medium. The yeast strain used this fermentation modeling is Candida pseudotropicalis ATCC 8619 as investigated by Ghally and Taweel. The fermentation medium used was raw cheese whey which is also the same as the one used by Ghally and Taweel [1]. Other conditions such as micro-organism cultivation and fermentation condition are as described by Ghally and Taweel [1]. The characteristic of the raw cheese whey is presented in Table 2.

Table 2. Raw cheese whey characteristics.

Characteristics Measured Values

Units

Total solids 68,300 mg/L Fixed solids 6,750 mg/L Volatile solids 61,550 mg/L Percent volatile solids 90.12 % Percent fixed solids 9.88 % Suspended solids 25,150 mg/L Total Kjehdahl nitrogen 1,560 mg/L Ammonium nitrogen 260 mg/L Organic nitrogen 1,300 mg/L Percent organic nitrogen 83.33 % Percent ammonium nitrogen 16.67 % Total COD 81,050 mg/L Soluble COD 68,050 mg/L Insoluble COD 13,000 mg/L Percent soluble COD 84.96 % Percent insoluble COD 16.04 % Lactose 4.82 % pH 4.90

Modeling. The mathematical model of continuous fermentation process of cheese whey by Candida pseudotropicalis has been derived as shown in Eqs. (2), (7), (12), (15). (16) and (17). The continuous fermentation is influenced by the value of hydraulic retention time (R). For the first reactor; at the certain value of R, the specific growth rate was determined using Eq. (7) and the concentration of remaining substrate, cells and ethanol produced can be evaluated by solving Eqs. (2), (12), and (15) simult-aneously. For the second reactor, all the

unknown variables were calculated by solving Eqs. (2), (12), (16) and (17) simultaneously.


The Profile of cell concentration and individual ethanol productivity are shown in Figures 2 and 5, respectively. As can be seen in Figure 3, there was certain amount of substrate that remained unconsumed by the cell at the outlet of the first reactor. Further processing in the second reactor would give more benefits, i.e. higher product concentration could be obtained (see Figure 4.) and lower substrate outlet concentration (see Figure 3 for Reactor 2). Lower substrate concentration is favored as it would not result in environmental problem. Previous results showed [4] that the recommended value of hydraulic retention time for the first reactor is 20 h as it resulted in the highest ethanol productivity. This result is shown in Table 3.

In this study, at 50 g/L first reactor inlet substrate concentration, the range of hydraulic retention time used in the second reactor is between 14-19 h. Figures 2 to 5 indicate that 14 h hydraulic retention time would give maximum cell, substrate and ethanol concentration as well as ethanol productivity.

20 25 30 35 40 45 50

0.2

0.25

0.3

R (Hydraulic retention Time) [h]

X [

g/L]

14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 190.2

0.25

0.3

0.35


X [

g/L]

First Reactor

Second Reactor

Figure 2. Cell concentration profile within the first and second reactor at Si = 50 g/L.



76

20 25 30 35 40 45 500

5

10

15

20


S [

g/L]

14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 190.1

0.2

0.3

0.4

0.5


S [

g/L]

First Reactor

Second Reactor

Figure 3. Substrate concentration profile within the first and second reactor at Si = 50 g/L.

20 25 30 35 40 45 501.5

2

2.5


P [

g/L]

14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 192.2

2.3

2.4

2.5


P [

g/L]

First Reactor

Second Reactor

Figure 4. Ethanol concentration profile within the first and second reactor at Si = 50 g/L.

20 25 30 35 40 45 500

0.05

0.1

0.15

0.2


Pro

duct

ivity

[g/

L.h]

14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 190.12

0.14

0.16

0.18


Pro

duct

ivity

[g/

L.h]

First Reactor

Second Reactor

Figure 5. Individual ethanol productivity for the first and second reactor at Si = 50 g/L.

Table 3. Recommended R, product concent-ration, and productivity for the first reactor.

Si Recommended R value

P1 [g/L]

Productivity [g/L.h]

50 20 2.1829 0.1091 100 20 6.3263 0.3163 150 20 11.2783 0.5639

Table 4 shows the recommended values of R, ethanol concentration and substrate concentration at three different inlet substrate concentrations. By using two reactors in series, it was found that the ethanol concentration only slightly increased compared to using single reactor. However, it resulted in significant reduction of substrate concentration.

Table 4. Summary of simulation results.

Si

Recommended R value Reactor 1 Reactor 2

Ethanol Concentration [g/L]

Substrate Concentration [g/L]

50 20 14 2.4681 0.3719 100 20 7 6.4755 0.1928 150 20 4 11.6858 0.7964



77

5. Conclusions

A steady state model for cheese whey fermentation in two series of reactors has been developed. Steady state simulation results for various hydraulic retention time and inlet substrate concentration indicated that the maximum ethanol productivity was achieved at low value of hydraulic retention time. The higher the inlet substrate concent-ration the higher the ethanol concentration

and productivity would be. For certain value of volumetric flow rate, the dimension of the second reactor will be smaller compared to the first reactor as the recommended value of R for the second reactor is smaller than the first. Combining two reactors in series for continuous cheese whey fermentation result-ed in significant reduction of substrate concentration and slightly higher ethanol concentration compared to the use of single reactor.

References

[1] Ghaly, A.E., & Taweel, A.A. (1994). Kinetic modeling of batch production of ethanol from cheese whey. Biomass and Bioenergy, 6, 6, 465-8.

[2] Ghaly, A.E., Taweel, A.A. (1997). Kinetic modeling of continuous production of ethanol from cheese whey. Biomass and Bioenergy, 12, 6, 461-72.

[3] Kargi, F., & Ozmihci, S., (2006). Utilization of cheese whey powder (CWP) for ethanol fermentations: effect of operating parameters. Enzyme and Microbial Technology, 38, 711-8.

[4] Agustriyanto, R., & Fatmawati, A. (2009). Model of Continuous Cheese Whey Ferment-ation by Candida pseudotropicalis. In Proceedings of. 57th World Congress of

Science, Engineering and Technology (WCSET), Amsterdam: WASET, pp. 281-5.

[5] Zafar, S., & Owais, M. (2005). Ethanol production from crude whey by Kluyverom-yces marxianus. Biochemical Engineering Journal, 27, 295-8.

[6] Friend, B.A., Cunningham, M.L., & Shahani, K.M. (1982). Industrial alcohol production via whey and grain fermentation. Agricult-ural Waste, 4, 55-63.

[7] Blanch, H.W., & Clark, D.S. (1997). BiochemicalEnginering. New York: Marcel Dekker Inc.

[8] Shuler, M.L., & Kargi, F. (1992). Bioprocess Engineering Basic Concept. New Jersey: Prentice-Hall.


SCIENCE JOURNAL Ubonratchathani University http://scjubu.sci.ubu.ac.th

Sci. J. UBU, Vol. 1, No. 2 (July –December, 2010) 78-83


Research Article

Image Structure of Large Dams in Kanchanaburi, Thailand, Using Surface GPR Reflection Techniques

S. Yooyuanyong*, W. Sripanya

Department of Mathematics, Faculty of Science, Silpakorn University, Nakhon Pathom 73000, Thailand.


1. Introduction

Fault is the name of situation that the rock was pressured to some direction and the rock is broken for a long distance. There are 5

kinds of faults; normal fault or gravity fault, reverse fault or thrust fault, strike-slip fault, oblique-slip fault, and rotation fault. There are 13 faults in Thailand; Mae Chan and Mae Ing Fault, Mae Hong Son Fault, Moei Fault, Mae Tha Fault, Thoen Fault, Phayao Fault, Pua Fault, Uttaradit Fault, Three Pagoda Fault, Sisawat Fault, Thakhaek Fault, Ranong

Abstract

Nowadays, earthquakes often take place around the world. The location of earthquakes actually locates close to the fault. There are two large faults locate in Kanchanaburi, Western of Thailand. The safety and operability of the large damp depends on the quality of material used to construct the body of the dam as well as to the earthquakes and fault in that area. It is known that the dam suffers from many different structural problems which can lead to damage to the body of dam, for instant; earthquakes, fault, the weight of car while it run on the pavement of the dam, the weight of dam and the weight of the water in the dam. Many forms of damage, originating in the bottom layers are invisible until the pavement or the body of the dam cracks. They depend on the infiltration of water and the presence of cohesive soil greatly reduces the bearing capacity of the cement or sub asphalt layers and underlying soils or rock. On the basis of an in-depth literature review, an experimental survey with Ground Penetrating Radar (GPR) was carried out. The experiments were set on a pavement on the top of the dam. GRP travel time data were used to transmit and receive the electromagnetic field and given the image of the medium into 2-dimensional cross section. The image figure results of sounding from Khaoleam Dam and Srinakarin Dam in Kanchanaburi province showed that there was no damage in the structure of dam. It means that, up to now, Si Sawat Fault and Three Pagoda Fault do not take effect to the body of both dams.

Keywords: Ground-Penetrating Radar (GPR), Srinakarin Dam, Khaoleam Dam, Si Sawat Fault, Three Pagoda Fault.

S. Yooyuanyong et al., Sci. J. UBU, Vol. 1, No. 2 (July –December, 2010) 78-83


79

Fault, and Khlong Marui Fault as shown in Figure 1 [1]. In this paper, we concentrate only 2 faults in the western of Thailand. Si Sawat Fault is the big one and still effective currently. The other fault is Three Pagoda Fault which is still effective currently as well. The report from Department of Metrological in Bangkok stated that there were 121 Earthquakes during 15 April 1983 to 19 June 1983 at Amphor Si Sawat, Kanchanaburi province. Three of them effected to the building in Bangkok during 15 to 22 of April 1983 [2]. These Earthquakes were happened after the construction of Khaoleam Dam and Srinakarin Dam. It was possible that the weight of water in the dam took effect to the structure of the ground. Unfortunately, there were faults pass to that region, thus, the base of the ground would rupture. It would effect to the body of the dam directly. Earthquakes in Thailand often happen in many areas. The people in Thailand do not so frighten since the situation is not serious. However, up to now, there are many huge building and particular many large dam, thus, we have to encourage the people to take care of them and pay attention to their security.

Kanchanaburi is the province which has long and huge mountain. There are two faults, namely, Si Sawat Fault and Three Pagoda Fault pass over the province. The Si Sawat Fault is 500 kilometers long pass under Mae Klong River and Kwai Yai River and the Three Pagoda Fault is 250 kilometers long pass under Kwai Noi River. Srinakarin Dam crosses over the Si Sawat Fault and Khaoleam Dam crosses over the Three Pagoda Fault. More than 7 Richters of Earthquakes may damage to both dams. To study the structure of dam, the knowledge in geophysics is very important. For humans, the shallow subsurface is perhaps the most important geological layer in the earth. This layer contains many of the earth’s natural resources (e.g. building aggregates/stone, placer deposits, drinking water aquifers, soils) and also acts as a sink for human waste (e.g. landfill sites). In addition, through the study of rocks and unconsolidated sediment accumulations at or near the surface we have

discovered much about earth history and behavior of its dynamic landforms. These insights have aided environmental manage-ment, such as prediction of natural disasters, helped exploration for more remote natural resources such as oil and gas, and increased understanding of the geological development of other planets in our solar system. Given the large rise in human population predicted

for the 21st

century, a more detailed understanding of the shallow subsurface will be required if humans are to sustainably manage many of the earth’s finite resources. As Grasmuck and Green [3] note, given the importance of the earth’s upper layers to human development, it is surprising that

during much of the 20th

century techniques for exploring them did not change significantly. Analysis of field exposures linked via data from limited numbers of widely spaced boreholes, shallow excavat-ions and geophysical surveys is still typical. Drilling and trial pits are time consuming and expensive, often yielding only limited additional information that is difficult to correlate between distant sampling points. In some instances such invasive techniques cannot be implemented due to environmental or conservation considerations. The most common geophysical techniques employed in shallow subsurface investigations are seismic reflection. Consequently, during the 1970s attention increasingly turned to using other, higher resolution, geophysical techniques. One technique that has proved extremely useful is ground-penetrating radar (GPR or ground-probing radar, surface penetrating radar, subsurface radar, georadar or impulse radar). GPR detects electrical discontinuities in the shallow subsurface (typically less than 50 m) by generation, transmission, propa-gation, reflection and reception of discrete pulses of high-frequency electromagnetic energy in the megahertz (MHz = 106 Hz, 1 Hz = 1 cycle/s) frequency range. GPR’s origins lie in research carried out during the

early 20th

century by German scientists trying to patent techniques to investigate the nature of various buried features [4,5].



80

Figure 1. Map of faults in Thailand.



81

Pulsed electromagnetic waves were first used in the middle of 1920s. Following these initial developments, much early work using radar was in glaciology [6], with civil engineering, archaeological and geological applications becoming more frequent from the 1970s onwards [4,5,7]. However, it was not until the 1980s that GPR systems became commercially available and digital data acquisition was feasible [8]. Due to GPR’s relatively recent development and accept-ance, and the wide range of potential uses, experience of GPR endusers is wide and their subject backgrounds diverse. The following of this section is, therefore, offered as a contribution to the direction of GPR research in structural damages of both Khaoleam Dam and Srinakarin Dam in Kanchanaburi, west of Thailand. Khaoleam Dam (Wachiralong-khorn Dam) is one of the biggest water reservoirs situated in Kanchanaburi, west of the country near the Burmese border. It is also a peaceful place that encourages leisure, adventure and unity with nature. The capacity of Khaoleam Dam is 5,848 million cubic meters of water and the pavement over the dam is 1,300 meters long. Srinakarin Dam is also located in the province of Kanchanaburi. It was constructed to provide a natural water and power source for the surrounding area. The capacity of Srinakarin Dam is 7,448 million cubic meters of water and the pavement over the dam is approximately 1,500 meters long.


In our study, the data were collected at Khaoleam Dam (Wachiralongkhorn Dam) and Srinakarin Dam by using ground penetrating radar (GPR). The electromagnetic field data including the time response from the ground were recorded by using ZONE 12 ground penetrating radar and the data were interpreted by using PRISM Software, ran on HP notebook computer, to get two dimens-ional graphs of XZ plane as shown in Figures 2 to 5 for Khaoleam Dam and figures 6 to 8 for Srinakarin Dam. The electromagnetic responses over the body of the dams can be used to show the image structure of

Khaoleam and Srinakarin Dam. The two dimensional image graphs were plotted to reveal the continuation of the material of the dam. The discontinuity of material of the dam body may imply to the dam suffers from many different structural problems which can lead to damage to the body of dam, for instant; earthquakes, fault, the weight of car while it run on the pavement of the dam, the weight of dam and the weight of the water in the dam. Many forms of damage, originating in the bottom layers are invisible until the pavement or the body of the dam cracks


The image graphs are shown in Figures 2 to 8. We changed the frequencies of electro-magnetic source and the graphs of image ground structures were performed to be accurated at some depths only. The vertically maximum depth that we could be able to investigate was 35 meters starting from pavement on the top of dam. Figures 2 to 5 were the image cross section in XZ plane of Khaoleam Dam. We used the frequencies of 38, 75, 100, and 300 MHz for sounding over the dam. The suitable frequencies to investigate Khaoleam Dam were 300, 700 and 900 MHz. The images Figures 2 to 5 showed that the ground structure from the

pavement horizontally on the top of the dam

to the base or bottom of the dam were

smooth. Figure 2 displayed the image of

Khaoleam Dam structure, using 38 MHz

antennae scan to collect data from West to

East direction. The vertical image direction

Figure 2. Khaoleam Dam structure, using 38 MHz antennae.



82

exposed the layered of the material used to construct the dam. We observed that the layered of material used to construct the dam were not smooth and were not parallel to the ground surface according to the weight of the material used and the engineering construct-ion process. Figure 3 showed the image structure of Khaoleam Dam, using 75 MHz antennae scan to collect data from West to East direction. The parabola curves in the figure exhibited the response of the electromagnetic field to the different media. The parabola area in the figure was the pipeused to control the water level of the dam. The diagonal straight line on the left hand side of the figure was represented to the junction of the cement. Figure 4 was similar

to Figure 3 that showed Khaoleam Dam’s structure, using 100 MHz antennae scan to collect data from West to East direction. Figure 5 displayed the image structure of Khaoleam Dam, using 300 MHz antennae scan to collect data from West to East direction. The dark regions in Figure 5 was represented the large vertical hold of the dam.

Figures 6 to 8 were the image cross section in XZ plane of Srinakarin Dam. We used the frequencies of 300, 700 and 900 MHz for sounding over the dam. The suitable frequencies to investigate Srinakarin Dam were 700 and 900 MHz which were different from the frequencies used at Khaolem Dam. The entire image Figures 6 to 8 revealed the ground structure from the pavement horizontally on the top of the dam to the base or bottom of the dam which was smooth.



Figure 6. Srinakarin Dam structure, using 300 MHz antennae.






83

Figure 6 exposed the image of Srinakarin Dam’s structure, using 300 MHz antennae scan to collect data. The vertical image showed the layered of the material used to construct the dam. It could be seen that the layered of material used to construct the dam were not smooth and were not parallel to the ground surface according to the weight of the material used and engineering construction process. Figure 7 showed the image of Srinakarin Dam structure, using 700 MHz antennae scan to collect data. The parabola curves in the figure showed the response of the electromagnetic field to the different media. The parabola area in the Figure 7 was the pipes used to control the water level of the dam. Figure 8 was similar to Figure 7 that displayed Srinakarin Dam structure, using 900 MHz antennae scan to collect data.

4. Conclusions

It could be concluded that the structure of Khaoleam and Srinakarin Dam were very good. The image figures showed that the body of the dams was perfectly continuous except for the junction of the cement used and the pipes to release the water. It did not have any damage regions. Up to May, 11, 2010, we could be able to say that Si Sawat fault and Three Pagoda fault did not take effect to Srinakarin Dam and Khaoleam Dam. In future, we have to repeat our work again to recheck our results and need to use different methods to confirm our results.

Acknowledgements

This research is supported by Faculty of Science, Silpakorn University and Centre of Excellence in Mathematics.

References

[1] Department of Mineral Resources (2010). http://www.dmr.go.th/ewtadmin/ewt/ dmr_web/main.php?filename=fault_En. [2] Society for the conservation of national treas- ure and environment (1983). Kanchanaburi Dam. Kanchanaburi: Kanchanaburi Publish- ing. [3] Grasmuck, M., & Green, A.G. (1996). 3-D geo radar mapping: looking into the subsurface. J. Environ. Eng. Geosci. 2, 195–200. [4] Daniels, D. J. (1996). Surface-penetrating Radar. Institute. Electric. Engineers, London : Cambridge Press. [5] Reynolds, J.M. (1997). An Introduction to

Applied and Environmental Geophysics. New

York : Wiley Chichester. [6] Plewes, L.A., & Hubbard, B. (2001). A review of the use of radio-echo sounding in glaciology. Prog. Phys. Geogr. 25, 203–36. [7] Conyers, L.B., & Goodman, D. (1997). Ground-Penetrating Radar: An Introduction for Archaeologists. London : Altamira Press. [8] Annan, A.P., & Davis, J.L., (1992). Design and development of a digital ground penetrating radar system. In: J. Pilon (Ed.), Ground acknowledgements penetrating radar. Geol. Surv. Can. Pap. 90, 15–23.

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