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Sources of Chitin and Chitosan and their Isolation

Leen Bastiaens, Lise Soetemans, Els D'Hondt, and Kathy Elst

VITO - (Flemish Institute for Technological Research), Mol, Belgium

Chitin is a natural biomolecule that was reported for the first time in 1811 by the French professor Henri Braconnot as fungine [1] and in 1823 by Antoine Odier as chitin. Chitin consists of large, crystalline nitrogen-containing polysaccharides made of chains of a modified glucose monosaccharide, being N-acetylglucosamine. It is ubiquitously present in the world and has even been reported to be one of the most abundant biomolecules on earth, with an estimated annual production of 1011-1014 tons [2, 3]. Chitin serves as template for biomineralization such as calcification and silicification, providing preferential sites for nucleation, and controlling the location and orientation of mineral phases [4, 5]. This phenomenon explains the presence of chitin in solid structures in a variety of biomass such as cell walls of fungi and diatoms and in exoskeletons of Crustaceans. Chitin is present in diverse structures in at least 19 animal phyla besides its presence in bacteria, fungi, and algae [5].

Chitosan is mainly known as a partially deacetylated derivative of chitin that is more water soluble than chitin, and as such is easier to process. For this reason, chitosan-and, in some cases, even more preferably, the relatively small sized (1-10 kDa) chitosan oligomers-are the molecules that are envisioned for multiple applications such as agriculture; water and wastewater treatment; food and beverages; chemicals; feed; cosmetics; and personal care [6, 7]. In addition, chitosan oligomers have been reported as being bioactive [8], offering potential for application in, for instance, wound dressing and cosmetics. Although chitin and chitosan are versatile and promising biomaterials [9], the extraction and purification of chitin and its conversion to chitosan (oligomers) require several process steps, and these have been mentioned as bottlenecks that hinder the wider use of the underspent chitin in the world.

This chapter intends to provide more information related to (1) the structure of chitin, (2) sources of chitin and chitosan, and (3) their extraction and purification, as well as (4) the conversion of chitin into chitosan. The further conversion of chitosan to chitosan oligomers is the subject of Chapter 3.

1.1 Chitin and Chitosan

1.1.1 Chemical Structure

Chitin, and its derivate chitosan, are natural polysaccharides consisting of 2 monosaccharides, N-acetyl-D-glucosamine and D-glucosamine, connected by ß-1,4- glycoside bonds. Depending on the frequency of the latter monosaccharides, the molecule is defined as chitin or chitosan. Chitin contains mainly N-acetyl-D-glucosamine and can be transformed to chitosan by partial deacetylation of the monomer N-acetyl-D-glucosamine to D-glucosamine (see Figure 1.1) [7]. Diverse definitions of chitin and chitosan circulate in literature. Most sources mention a deacetylation degree of at least 50% [7, 10] as a criterion to define the molecule as chitosan. Others report a deacetylation degree of at least 60% or 75% for chitosan, implying that, respectively, more than 60% or 75% of the monosaccharides are D-glucosamine moieties [11-13]. Chitin in its natural appearance is usually already a heteropolymer, with a deacetylation degree ranging between 5% and 20% [14]. The structure of chitin is very similar to that of cellulose and shares generally the same function of providing structure integrity and protection of the organism.

1.1.2 Different Crystalline Forms of Chitin

Chitin usually functions as a supporting material and is composed of layers of polysaccharide sheets. Each individual sheet consists of multiple parallel-positioned chitin chains [17], as schematically presented in Figure 1.2. Highly crystalline fibers are formed when the polymer sheets are placed next to each other and form interactions [12]. Depending on their orientation, three crystalline forms have been reported (a, ß, and ?).

The most abundant form is a-chitin, which is present in almost all crustaceans, insects, fungi, and yeast cell walls [7]. In this formation, the chitin sheets (three sheets as example in Figure 1.2a), consisting of parallel chitin chains (for each sheet, two chains are presented in Figure 1.2a), are positioned in an anti-parallel way, allowing a maximum formation of hydrogen bonding. More specifically, two intramolecular and two intermolecular bondings are formed: a first intermolecular bonding with a vertical neighbor chain (in the same sheet), and another with a horizontal neighbor chain form a different sheet [15]. These hydrogen bounds create a remarkably high crystallinity, resulting in a more stiff and stable material. Therefore, a-chitin is characterized as a non-reactive and insoluble product [16]. Since this form is the most common polymorphic, a-chitin has been extensively studied [12].

On the other hand, in ß-chitin, the chitin sheets are ordered in parallel (Figure 1.2b) with weaker intermolecular forces. This results in a softer molecule with a higher affinity for solvents and a higher reactivity. It is proven to be soluble in formic acid and can be swollen in water [15]. This chitin form is present in the squid pen, in the tubes of pogonophoran and vestimentiferan worms, and in monocrystalline spines excreted by diatoms such as Thalassiosira fluviatilis [7]. Although squid and tubes of Tevnia jerichonana both contain ß-chitin, their crystallinity differs. This implies that the crystallinity also depends on the source. Chitin obtained from squid pens is semi-crystalline, and chitin from T. jerichonana is almost complete crystalline [7, 8, 16].

Figure 1.1 Chemical structure of chitin and chitosan and some examples of species that contain chitin.

Figure 1.2 Schematic representation of (a) a-form and (b) ß-form of chitin.

The third formation, ?-chitin, is less common. It is considered to be a mixture or intermediate form of a- and ß-chitin with both parallel and antiparallel arrangements [16]. More specifically, every third chitin chain has the opposite direction to the two preceding chitin sheets [13, 15]. Very few studies have been carried out on ?-chitin, and it has been suggested that ?-chitin may be a distorted version of the other two instead of a true third polymorphic form.

1.2 Sources of Chitin and Chitosan

1.2.1 Sources of Chitin

For more than a century, scientists reported chitin to be present in a variety of organisms. Initially, zoologists named all hard yellow-brownish structures chitin, without chemical analysis, sometimes generating misleading data. Later on, it was accepted that the presence of chitin could only be demonstrated after chemical tests. Hymann (1958), for instance, used an iodine-based color test to study the presence of chitin in different sea animals. Later on, more sophisticated techniques such as Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), mass spectroscopy (MS), X-ray diffraction (XRD), and Raman spectroscopy were used [18]. Quantification of chitin is challenging and only reported in more recent publications. Currently, quantitative data on chitin contents are still incomplete, and available numbers need to be interpreted with care. Not only are different quantification methods used, but also varying parts of the biomass are considered (whole organism versus chitin-rich part of the organisms).

Nowadays, it is estimated that a large portion of chitin produced in the biosphere is present in the oceans [19, 20]. It can be found in aquatic species belonging to phyla such as Cnidaria (corals [21, 22]), Entoprocta [23], Phoronida (horseshoe worms [18]), Ectoprocta [18], Brachiopoda (lamp shells [18]), Bryozoa [19], Porifera (sponges [5, 24]), and Mollusca (squid [8, 23], cuttlefish [26], and clams [8]). Further, chitin has also been detected in fungi (mushrooms and yeasts [1]), algae (diatoms [27], coralline algae [28], green algae [29, 30]), Onychophora (velvet worms), and protozoa [31]. The most easily accessible sources of chitin, however, are the exoskeletons of Arthropoda, which includes insects [32-35], arachnids (spiders [36] and scorpions [37]), myriapods (millipedes and centipedes [38]), as well as Crustaceans (shrimp, krill, crab, and lobster [8, 9, 18, 37]).

Table 1.1 lists examples of chitin-containing sources, along with available compositional data. The amount of chitin varies with species type, the biomass part considered, and even with seasons and growth stages [40]. Values ranging from...