Chitin, poly (b-(1-4)-N-acetyl-D-glucosamine), is a natural polysaccharide of major importance. It was first discovered by Braconnot (1811), a professor of natural history. He isolated chitin from mushrooms by treating it with warm alkali. Later Odier (1823)Â found chitin while studying beetle cuticles and named "chitin" after Greek word "chiton" (tunic, envelope). The silk worm was also discovered as a source of chitin when Lassaigne (1843) isolated it from the Bombyx mori. The monomeric unit of chitin (N-acetyl glucosamine) became known because of the work of Ledderhose in 1878. In the first half of the twentieth century, research on chitin was mostly directed toward the study of its occurrence in living organisms. Finally in 1981 Austin and his coworkers came up with a completed data on the sources of chitin which is widely distributed in marine invertebrates (Figure 1), insects, fungi, and yeast (1981). However, chitin is not present in higher plants and higher animals. Generally, the shell of selected crustacean was reported by Knorr (1984) to consist of 30-40% protein, 30-50% calcium carbonate and calcium phosphate, and 20-30% chitin. Chitin is widely available from a variety of source among which, the principal source is shellfish waste such as shrimps, crabs, and crawfish (Allan et al., 1979). It also exists naturally in a few species of fungi.
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Chitin occurs in nature as ordered crystalline microfibrils forming structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast. It is also produced by a number of other living organisms in the lower plant and animal kingdoms, serving in many functions where reinforcement and strength are required. (Rinaudo, 2006). The structure of chitin has been described (Fig. 1). In terms of its structure, chitin is associated with proteins and, therefore, high in protein contents. Chitin fibrils are embedded in a matrix of calcium carbonate and protein. The matrix is proteinaceous, where the protein is hardened by a tanning process (Muzzarrelli, 1977). Studies of Ashford et al., (1977) demonstrated that chitin represents 14-27% and 13-15% of the dry weight of shrimp and crab processing wastes, respectively.
Fig. 1. Seafood Produces Considerable Amounts of Waste which are the Principal Source
2.2. Characteristics and Structure of Chitin
Chitin is made up of highly extended hydrogen bonded chain and is semi-crystalline in structure of chitin [Rinaudo(2006)& Kurita (2001)]. Chitin is a structural biopolymer, which has a role analogous to that of collagen in the higher animals and cellulose in terrestrial plants [Muzzarelli, (1977) & Mayer, (1996)]. Plants produce cellulose in their cell walls and insects and crustaceans produce chitin in their shells (Muzzarelli, 1986). Cellulose and chitin are, thus, two important and structurally related polysaccharides that provide structural integrity and protection to plants and animals, respectively [Muzzarelli (1986) and Roberts(1992)]. Chitin occurs in nature as ordered crystalline microfibrils forming structural components in the exoskeleton of arthropods or in the cell walls of fungi and yeast (Raabe 2007). In crustaceans, Chitin polymers tend to form rod like fibrils or crystallites that are balanced by hydrogen bonds formed between the amine and carbonyl groups.
X-ray diffraction analysis suggests that chitin is a polymorphic substance that occurs in three different crystalline modifications, termed Î±, ß and Î³ chitin. They mainly differ in the degree of hydration, in the size of the unit cell and in the number of chitin chains per unit cell [Rudall and Kenchington, (1973) Kramer and Koga, (1986)]. In the Î± form, all chains exhibit an anti-parallel orientation; in the ß form the chains are arranged in a parallel manner; in the Î³ form sets of two parallel strands alternate with single anti-parallel strands. Chitin is found to occur as fibrous material embedded in a six stranded protein helix [http://meyersgroup.ucsd.edu, 2006]. The polymorphic forms of chitin differ in the packing and polarities of adjacent chains in successive sheets; in the termed Î± form, all chains are aligned in a parallel manner, which is not the case in ß form and Î³ chitin. The molecular order of chitin depends on the physiological role and tissue characteristics. In both structures, the chitin chains are organized in sheets where they are tightly held by a number of intra-sheet hydrogen bonds with the ß- and Î³ chains packed in antiparallel arrangements Rinaudo. (2008).
Fig.2. Structure of Cellulose and Chitin
2.3. Biodegradation of tiger prawn shell by Lactic acid fermentation for extraction of Chitin
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Every year tones of sea food waste is dumped onto the shores of the sea and lagoons or in the inner mangrove area surrounding the sea for these are the regions where maximum sea food cultivation is done. These areas are the hub of number of small and large scale seafood industries which deal with culturing and processing of seafood. This huge amount of sea food waste is polluting the surrounding land and water and is depleting the fresh water supply. Dumping of Seafood waste leads to accumulation of sediments causing organic pollution which causes physical disturbance of hydrological regimes resulting in a number of ecological problems which include conversion and degradation of costal ecosystem. ( Mathew and Nair, 2006)
The demineralization of crustacean shells have been chemically performed using concentrated acids such as HCl (Whistler et al., 1962), H2SO4 (Peniston and Johnson, 1978), CH3COOH (Bautisa et al., 2000) and HCOOH (Horowitz et al., 1957) by various researchers. However, the chemical methods are expensive and detrimental to the environment leading to effluent problems [Shirai (2001) and Fagberno (1996)]. The Traditional method of chitin preparation from crustacean waste involving the use of alkalis and acids for demineralization, make the method ecologically harsh and a cause of pollution (Rao et al., 2000)
It also reduces the chitin quality to certain extent (Simpson et al. 1994; Healy et al., 1994) mostly such processes depolymerising chitin to a higher extent leading to the formation of a deacetylated form of chitin called chitosan.
Washing and drying
Grinding and sieving
3.5% NaOH (w/v) for 2 h at 65oC, solid: solvent (1:10, w/v)â†“
1 N HCl for 30 min at room temp., solid: solvent (1:15, w/v)â†“
Washing and Drying
Fig. 3. Traditional chitin Production Flow Scheme (No and Meyers, 1995)
Biotechnological process of lactic acid fermentation of crustacean shell waste is a powerful tool to overcome the environmental problems. Fermentation of crustacean shells using lactic acid bacteria is also an attractive method which lowers the pH of the medium and facilitates the demineralization of minerals and the hydrolysis of proteins while leaving the associated chitin intact. Thus this process also helps in a safe recovery of chitin as the fermented residue. Also, fermentation of crustacean bio waste to recover chitin considerably replaces the expensive and non environmentally friendly chemical process [ Rao et al., (2000), Shirai et al., (2001) and Hall et al., (1992) ].
Lactic acid bacterial fermentation of shrimp waste for chitin recovery was studied with lactose or cassava extract as additional sources of carbohydrate for natural energy (Hall and Silva 1992). Raw heads of Africa river prawn were fermented with Lactobacillus plantarum using cane molasses (Fagbenro 1996). Treatment of minced waste of scampi in the presence of glucose by a culture of Lactobacillus paracasei strain A3 was investigated (Zakaria et al. 1998). The primary object of all these studies was demineralization of the raw materials along with which deproteinisation took place (Shirai et al. 2001). The effectiveness of demineralization was exaggerated by the increasing inoculum amounts supplied. Also, the proportion of glucose was significant for the lactic acid fermentation by the bacterial strain to demineralize the shell wastes (Shirai et al. 2001 and Rao et al. 2002).
The demineralized and deproteinized chitin has a light pink color due to the presence of astaxanthin pigment. When bleached product is desired, this pigment can be eliminated by the decolorization using bleaching agents. The resulting chitin is insoluble in most organic solvents; however, its deacetylated derivative chitosan is soluble in some acids. The subsequent conversion of chitin to chitosan is generally achieved by treatment with concentrated sodium hydroxide solution (40-50%) at 100°C or higher for 30 minutes to remove some or all of the acetyl groups from the polymer (No and Meyers, 1995).
Lactic acid bacterial fermentation for demineralization has also been occasionally reported for shrimp waste (Shirai et al. 2001) crayfish exoskeleton (Bautista et al. 2001) and scampi waste (Zakaria et al. 1998). However, demineralization by lactic acid fermentation of tiger prawn shell waste along with the characterization of the resulting chitin has been less studied in relation to glucose concentration and inoculum amount. In the present work, we evaluated the demineralization of tiger prawn shell waste by lactic acid bacterial fermentation with various concentrations of inoculum and glucose and characterize the fermented residue the chitin by powerful techniques such as X-Ray diffraction, FTIR, SEM and TGA.
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From the literature it is evident that the limitations of the chemical method for the degradation of sea food can be largely overcome by the biological method of demineralization and hence research interest has been shown in recent years in this direction. Lactic acid fermentation of crustaceans shell waste has been reported to be studied as a potential biological method of degradation (P Mathew and KGR. Nair, 2006)
2.4. Factors Affecting Production of Chitin by Lactic Acid Fermentation
2.4.1. Effect of Initial Glucose Concentration and Inoculation Level of Lactic Acid Bacteria on Tiger Prawn Shell Waste Fermentation
Amount of starter culture and initial glucose concentration are critical factors in the fermentation of tiger prawn shell waste fermentation. A correct proportion of initial glucose and starter culture concentrations increase the amount of lactic acid produced and thus increased the % demineralization. Glucose is a readily fermentable sugar and hence chosen as the source of carbon for the microbes in most of the studies. Glucose concentration is a highly important parameter of fermentation and hence chitin production. According to Jung et al. (2004) Microbial growth and hence acidification of the broth during fermentation is highly dependent on glucose concentration.
Lactobacillus sp. has the potential to produce lactic acid and other organic acids. Using organic acids such as lactic and/or acetic acids for the demineralization process is a promising idea since organic acids in order to produce low cost biomass, purified chitin and reduce the harmful to the environment (Jung et al., 2005,Rao et al., 2000, Sunita et al.,2009). According to Hong et al. (1999) the production of organic acids by the lactic acid bacterium L. plantarum decreased the pH and made the environment selective against spoilage microorganisms. Zakaria et al. (1998) had also reported that the decaying of the raw crustacean waste materials can be controlled through the selection of microorganisms having a high capacity to produce organic acids. Further Shirai et al. (2001) reported that the selection of the correct micro organism is an important factor for the acidification of crab shell waste and for suppressing the growth of spoilage organisms.
Cira et al., (2002) reported that lactic acid bacteria fermentation with the 10% inoculums was helpful in attaining a pH of around pH 5 after day 3. On the other hand it was reported by Shirai et al. (2001) that lactic acid fermentation of shrimp wastes which contained 10% glucose and a 5% inoculum of Latobacillus sp. B2 lowered from to pH 4.5. Therefore medium pH likely depends on the content of the energy source such as glucose and sucrose and the other factor least considered but of great importance is the solid to liquid ratio. Lower the solid to liquid ratio higher is the rate of demineralization. As the solid concentration increases the concentration of slurry increases resulting in reduced mass transfer and hence poor demineralization occurs. (Kyung. et al., 2008). The selection of the potential microbe along with the correct proportion of the additional starter is very important for the lactic acid bacterial fermentation to demineralize the raw shell wastes (Shirai et al. 2001; Rao et al. 2002) along with the correct propotion of solid to liquid ratio (Kyung.et.al. 2008).
2.4.2. Temperature of Fermentation
Application of microorganisms or enzymes to extract chitin from marine crustacean wastes is a current research trend for bio-conversion of wastes into useful biomass (Bhaskar et al., 2006). From his study he analyzed that a temperature of 35Â°C resulted in lowest pH conditions of pH 3.7 and highest % demineralization of about 92%. Kyung et al., (2008) reported that a temperature of 30Â°C gave the highest % demineralizatuion.
2.4.3. Particle Size
Particle size in chitin productions has sparked controversial reports on its effect on chitin quality. Some agree that small particle size is better than large particle size. According to Bough et al. (1978), smaller particle size (1mm) results in higher demineralization % with a chitin product of both higher viscosity and molecular weight than that of larger particle size (above 2 to 6.4 mm). The larger particle sizes require longer swelling time resulting in a slower deacetylation rate.
2.5. Process Optimization by Taguchi
Taguchi method of production optimization is a purely statistical approach to analyze scientific data based on statistical factorials. Taguchi experimental design offers remarkable advantages by examining a group of factors simultaneously and extracting as much quantitative information as can be extracted with a few experimental trials [Stone and Veevers, (1994) and Houng et al., 2006]. But yet only a few reports are available on the application of Taguchi's method in the field of biotechnology (Cobb and Clarkson, 1994 and Han et al., 1998).
2.6. Characterization and Physiochemical study of Chitin
2.6.1. X-Ray Diffraction Analysis
The crystalline structures of chitin are differently presented according to the raw materials. XRD is low cost and user friendly method to accurately characterize the kind of chitin extracted from a particular species. Chitin has three different crystalline polymorphic forms according to the derived material Î± chitin, Î² chitin, and Î³ chitin. The structures of the Î± and Î² forms differ only in that the piles of chains are arranged alternately antiparallel in Î± chitin, whereas they are all parallel in Î² chitin. The structures of Î± chitin, Î² chitin, Sugiyama et al., (1999) and Syed et al., 1999; have been determined by X-ray diffraction (XRD). According to the crystalline structure of chitin suggested by Rudall (1963) and (1967.) Î± chitin has strong intersheet and intrasheet hydrogen bonding,and Î² chitin chitin has weak hydrogen bonding by intrasheets. Therefore, in contrast to Î± chitin, Î² chitin is characterized by a weak intermolecular force, Lee et al., 1996. Not much information is available regarding the crystalline study of Î³ chitin by X ray diffraction technique. The XRD profiles of chitin samples easily help to distinguish the different forms of chitin based on the peaks and crystallinity. It has been found that Î± chitin has three to four sharp crystalline reflections at 9.6, 19.6, 21.1, and 23.7Â° whereas Î² chitin , has two broad crystalline reflections at 9.1 and 20.3Â° within the 2Î¸ range of 5-35Â°. These results also support that the crystallinity of Î² chitin is less than that of Î± chitin because of the parallel structure. Î± chitin has a more rigid crystalline structure because of its intersheets and intrasheets, and its structure exists as a stable structure with neither a crystalline phase transition nor thermal decomposition ( Jang et al., 2004).
2.6.2. FTIR Spectrophotometer Measurements
Different methods have been used for the purpose of measuring the degree of deacetylation of chitin for eg. the linear potentiometric titration, ninhydrin test, hydrogen bromide titrimetry, near-infrared spectroscopy, nuclear magnetic resonance spectroscopy, infrared spectroscopy, and first derivative UV-spectrophotometry. Among all the tests stated above FTIR is one of the potential methods to determine the degree of deacetylation of the sample. It is far easier yet highly sensitive compared to the other processes. The process of removal of acetyl groups from the molecular chain of chitin is called deacetylation, it leaves behind a high degree chemical reactive amino group (-NH2). Thus the physicochemical properties of chitin highly depend on the degree of deacetylation (DD) hence it determines its appropriate applications. (Khan et al., 2002) Degree of deeacetylation also affects the biodegradability and immunological activity (Tolaimate et al., 2003). The degree of deacetylation can also be used to differentiate between chitin and chitosan because it helps to know the amount of free amino groups in the polysaccharides. A degree of deacetylation of 75% or above in Chitin is generally known as chitosan (Knaul et al., 1999).
Fig.4- Structure of Chitin and Chitosan
The following formula by Domszy and Roberts (1985) is highly used for calculating the degree of deacetylation.
DD = 100 - [(A1655 / A3450) X 100 / 1.33]
The thermal degradation of chitin or chitosan with a broad range of DD has received little attention (Guinesi&Cavalheiro, 2006; Kittur, Prashanth, Sankar, & Tharanathan, 2002). There are fewer reports on the thermal degradation process of chitin/chitosan and its derivatives than on chemical and enzymatic degradation (De Britto & Campana-Filho, 2004; Holme, Foros, Pettersen, Dornish, & Smidsrod, 2001; Hong et al., 2007; Neto et al., 2005; Qu, Wirsen, & Albertsson, 2000; Wanjin, Cunxin, & Donghua,2005). Thus to examine the thermal degradation of chitin with a broad range of DD, thermogravimetric analysis (TGA) is a highly useful technique. It has also been reported that with an increase in the rate of deacetylation the temperature of degradation decreases (Young et al., 2009).
2.7. Application of Chitin
Chitin and chitosan has several distinctive biological properties, including biocompatibility and biodegradability, cellularbinding capability, acceleration of wound healing, hemostatic properties, and anti-bacterial properties (Cho, Cho, Chung, Yoo, & Ko, 1999; Muzzarelli, 1993; Tomihata & Ikada, 1997).Some of the important industrial applications of chitin have been listed below in Table 1.
Table1. Different industrial applications of chitin
Waste Water Treatment
Removal of metal ions, flocculant/coagulant, protein, dye
Thickener and gelling agent, animal feed additive.
Wound and bone healing, blood cholesterol control, skin burn
Seed Coat, Fertiliser, Controlled agrochemical release.
Moisturizer, face, hand, and body creams, bath lotion, etc
Enzyme immobilization, protein separation, cell recovery.