Chitin, Chitosan, and Alginate Fibers

Keywords

Chitin • Chitin dissolution • Xanthate • Chloroalcohol • Chitin-blend fiber • Wet stability • Fiber crosslinking

Several attempts were made in the 1920s and the 1930s to dissolve chitin using ionic salts and produce fibers. For instance, chitin was dissolved in lithium thiocy­anate and made into fibers. Instead of using native chitin, chitin xanthates were made by steeping chitin in 28-50 % NaOH solutions at room temperature for 2 h. Later the chitin was exposed to carbon disulfide to obtain the chitin xanthate. Fibers obtained were drawn to 250 % using hot glycerin. Dry strength of the fibers ranged from 1 to 1.2 g per denier, and the breaking elongation was 30 %. Using similar methods, other researchers had reported obtaining fibers with strength ranging from 0.9 to 1.5 g per denier with fineness being 3.08-18 deniers. Attempts were also made to combine chitosan xanthates with cellulose xanthates and produce fibers with better quality than chitin and viscose rayon fibers [97Agb]. However, fibers obtained using these approaches were considerably weak when wet and practically not useful. Some attempts had been made to cross-link the fibers with formaldehyde to improve wet stability, but the elongation and modulus had to be sacrificed [77Nog]. Some of the approaches used to obtain chitin fibers and the properties of the fibers obtained are listed in Table 24.1. Although good tensile strength was obtained using both the halogenated and amide-lithium chloride systems, the wet strengths were only about 0.2-0.5 g/denier, and the fibers were therefore not practically useful. Tokura et al. developed chitin fibers by suspending chitin in 99 % acetic acid to form a gel and later by dispersing the gel in dichloroacetic acid and isopropyl alcohol [79Tok]. Fibers obtained had fineness ranging from 2 to 25 denier, dry strength was between 0.7 and 1.6 g per denier, and elongation ranged from 2.7 to 3.4 %. However, the wet strength of the fibers was only between 0.1 and 0.3 g per denier.

Austin has listed several chloroalcohols that could dissolve chitin and produce fibers. Some of the alcohols listed include 2-chloroethanol, 1-chloro-2-propanol, 2-chloro-1-propanol, and 3-chloro-1,2-propanediol [75Aus]. Instead of using chitin in its native form, several researchers have produced chitin fibers by converting chitin in chitin xanthate. Filaments with tenacities of 0.6-1 g per denier were reportedly produced using the chitin xanthate. Recently, chitin was extracted from crustacean shells using ionic liquids, and the extracted chitin was directly used to produce chitin fibers [10Qin]. 1-Ethyl-3-methyl-imidazolium acetate was used to dissolve shrimp shells, and the dissolved solution was used to directly produce fibers. Fibers with ultimate stress of 1.58 g/den and elongation of 13 % were obtained using this approach.

Native chitin from krill was esterified with butyric acid to prepare dibutyrylchitin (DBCH) [06Bin]. The DBCH (15-25 %) was then dissolved in anhydrous ethanol and heated to 50-75 °C and then extruded into a coagulation bath. Fibers obtained were treated with KOH at various temperatures to deacetylate the fibers to various degrees. Deacetylation ranging from 3.8 to 70 % was obtained by varying the posttreatment conditions. It was also observed that treating with KOH converted the chitin into regenerated chitin fibers with a degree of crystallin­ity of about 76 %. Morphologically, treating the fibers with KOH resulted in the formation of micropores on the fiber surface.

Similar to blending chitin xanthates with cellulose xanthates, novel chitin (N — acetylchitosan)-silk fibroin blend fibers were prepared by Hirano et al., and the properties of the fibers were studied [02Hir]. Chitosan fibers made using 2 % acetic acid were N-acetylated using acetic anhydride. The chitin fibers were later dissolved using 14 % NaOH and mixed with various ratios of silk fibroin dissolved in lithium bromide. Fibers were extruded into a coagulation bath consisting of aqueous 10 % sulfuric acid saturated with ammonium sulfate. Subsequent treatments were done to remove the solvents and to complete the N-acetylation. Table 24.2 shows some of the properties of the fibers prepared. As seen from the table, adding fibroin decreased the strength and elongation of the fibers substan­tially due to the incompatibility between the protein and carbohydrate.

Fig. 24.2 Digital image of a knitted sock developed from chitin-fibroin (6 %) blend fibers. From [02Hir]. Reproduced with permission from Elsevier

Morphologically, the fibers had striations and a rough surface as seen from Fig. 24.1. Fibers containing 6 % silk fibroin were knitted into a sock shown in Fig. 24.2.

Although attempts have been made to produce fibers from chitin, difficulties in dissolving chitin have restricted the commercialization of the process. However,

chitin derivatized as chitosan is soluble in acetic acid, and extensive studies have been done on developing fibers from chitosan and are discussed in Chap. 25.

References

[75Aus] Austin: US patent 3879377

[77Nog] Noguchi, J., Tokura, S., Nishi, N.: Proceedings of the first international conference on chitin and chitosan, Boston, MA, p. 315 (1977)

[79Tok] Tokura, S., Nishi, N., Noguchi, J.: Polym. J. 11(10), 781 (1979)

[83Uni] Unitika Co. Ltd.: Japanese Patent # 58214513

[84Uni] Unitika Co. Ltd.: Japanese Patent # 59068347

[90Kif] Kifune, K., Inome, K., Mori, S.: US Patent 7932404

[97Agb] Agboh, O. C., Qin, Y.: Polym. Adv. Technol. 8, 355 (1997)

[02Hir] Hirano, S., Nakahira, T., Zhang, M., Nakagawa, M., Yoshikawa, M., Midorikawa, T.: Carbohydr. Polym. 47, 121 (2002)

[06Bin] Binias, D., Boryniec, S., Binias, W., Wlochowicz, A.: Fibres Text. East. Eur. 14(3), 12 (2006)

[10Qin] Qin, Y., Lu, X., Sun, N., Rogers, R. D.: Green Chem. 12, 968 (2010)

Regenerated Protein Fibers

Keywords

Recombinant protein • Protein expression • E. coli • Yeast • Transgenic plant • Yield • Molecular weight • Recombinant protein • Protein production • B. mori • Silkworm

Natural protein fibers such as spider silks have extraordinary properties, but it is difficult and impractical to obtain quantities of spider silk required for applications through the natural spinning process. To overcome this limitation, extensive efforts have been made to produce regenerated spider silk proteins using biotechnological approaches. Several heterologous host systems such as bacteria, yeast, mammalian cells, and transgenic plants, animals, and insects have been used to produce spider silk proteins as seen in Table 50.1 [12Chu]. Tokareva et al. provide a thorough review of the approaches used to produce recombinant spider silks and the limitations of the approaches [13Tok]. One of the most common and easiest approaches to obtain artificial spider silk is through bacterial production [07Ven]. Several researchers have expressed spider silk genes in Escherichia coli and have studied the structure, properties, and functions of protein fibers. Although bacterial production of proteins is possible on an industrial scale, several limitations have been expressed for this approach. The size of the expressible gene in E. coli is considerably smaller than the native gene found in spiders, and the bacteria use a distinct codon different than that in spiders. In addition, bacteria often remove repetitive sequences that are necessary to obtain the properties seen in spider silk fibers. To overcome these limitations, engineered genes that include the bacterial codon have been developed and expressed in E. coli. In one approach, artificial genes that encode the analogs of the proteins (spidroins 1 and 2) found in Nephila clavipes dragline silk were expressed in E. coli [97Fah1]. Proteins with purity of up to 99 % were obtained, and both the spidroins had mostly random structures.

In a unique approach, spider silk proteins were expressed in tobacco and potato plants using gene synthesis [01Sch]. Up to about 3 % of the total soluble protein

was accumulated in the plants. Proteins were extracted from the plants using a buffer and heating at 95 °C for 10 min. Fractional ammonium sulfate precipitation was used to further enrich the silk proteins. This system of production of proteins from plants was reported to be at least 50 % cheaper than producing proteins using bacterial systems. Recent studies have shown that transglutamination produced spider silk proteins in transgenic plants with elastic modulus similar to that of native spider silk [14Wei]. In addition to bacteria and plants, spider silk proteins have also been expressed in yeast such as Pichia pastoris [97Fah2]. It was found that the yeast was able to produce proteins with higher molecular mass with 3,000 amino acids similar to those found in natural proteins. The amounts of proteins produced were at least twice than that produced in bacteria. Table 50.2 lists the amount of cDNA yield obtained from various hosts when different spider silk genes were expressed. As seen from the table, a wide range of yields are obtained depending on the host and expression genes used.

Since dragline silk and flagelliform silk have highly distinct and unique properties, an attempt was made to engineer proteins that contained motifs from both the type of silks. Dragline silk is characterized by high strength of 35 g/den (4 GPa) but relatively low elongation (35 %) compared to strength of 8.7 g/den (1 GPa) but extensibility of 200 % for flagelliform silks [12Teu1]. In terms of structure, dragline silk consists of p-sheets forming domains with (GGX)n and/or (GPGXX)n motifs compared to the single and large protein (Flag) with (GPGGX) motifs held between shorter (GGX)n spacer motifs. The high strength of the dragline silk is due to the (aniline)n or (GA)n motifs that form p-sheet nanocrystals and preferentially align parallel to the fiber axis [12Teu1], whereas the extensibility of spider silks is due to the GPGXX repeat sequence [08Bro]. Two recombinant proteins with chimeric silklike sequences from dragline and flagelliform silks were engineered and cloned in E. coli. Proteins obtained had a molecular weight of 62 kDa (YIS820) and 58 kDa (AIS820). These proteins were lyophilized and dissolved in HFIP to obtain 26-27 % solutions and extruded into 90 % isopropyl alcohol and 10 % water coagulation bath. Fibers were drawn or further treated to improve mechanical properties. AIS820 could be directly spun into fibers, whereas YIS820 formed films, and fibers were hand drawn from the films. Table 50.3 provides a comparison of the tensile properties of the two types of proteins obtained. As seen from the table, AIS820 produced fibers with better tensile properties. It was suggested that the higher strength of the AIS820 fibers was due to the higher amounts of p-sheets that could be well aligned during fiber production. Both the fibers obtained were considerably weaker than their native form which was mainly attributed to the lower (1/5) molecular weight of the proteins used for regeneration of the fibers [12Teu1].

Several researchers have also considered expressing spider silk genes in the domesticated silkworm (Bombyx mori) with a view to obtain large quantities in a relatively short time [05Mot]. Spider silk genes were linked to enhanced green fluorescent protein (EGFP) and cloned and expressed in B. mori silks and larvae

[12Teu2]. It was reported that recombinant protein formed about 5 % of the total proteins in the cells. However, the silk obtained had low solubility and did not form fibers like spiders due to the aggregation of more than 60 % of the fusion proteins. To avoid the formation of aggregates, piggyback transformation vector containing spider genes (MaSp1 sequence) was introduced into silkworm eggs [12Teu2]. Although the fibers and cocoons containing spider silk were obtained, the mechani­cal properties of the fibers obtained were similar to that of B. mori silk but considerably lower than the native spider silk. In a similar approach, transgenic silkworms containing the spider silk gene A2S814 (78 kDa) and fibroin heavy chain from B. mori were expressed in B. mori silkworm eggs using piggyBac vectors. Enhanced green fluorescent protein (EGFP) was also included to enable easy detection of the combined genes. The eggs were incubated, hatched, and later reared in an incubator. Cocoons formed were collected from different generations of the transgenic silkworms, and fibers were extracted for analysis [12Teu2]. Figure 50.1 shows the images of the chimeric silkworm/spider silk/EGFP proteins

in cocoons, silk glands, and fibers obtained from the transformed cocoons. About 2­5 % of the composite protein was detected in the silk fibers. Table 50.4 provides a comparison of the properties of the silk fibers obtained from the transgenic silkworms with that of the properties of the native spider silks. As seen from the table, the composite fibers obtained from the silkworms had much higher tensile properties than the B. mori silks and toughness similar to that of the native spider silk fibers. Tensile strength and elongation of the composite fibers were much lower than that of the native spider silks. However, it should be noted that the fibers were tested at a humidity of 19-22 %, much lower than the standard humidity of 65 % used for testing textile fibers. Higher humidities could adversely impact the fiber properties.

Most of the approaches of reproducing spider genes in recombinant proteins have used small size amino acid sequences and molecular weights less than 120 kDa. However, the native spider silk proteins have molecular weights between 250 and 366 kDa, and therefore, it has not been possible to obtain regenerated spider silk with properties close to that of the native spider silk fibers [13Lin]. To overcome this limitation, a 248.9 kDa recombinant protein from the spider N. clavipes was expressed in metabolically engineered E. coli [10Xia]. Proteins that contained about 43-45 % glycine were made into fibers with tenacity of

4.4 g/den, elongation of 15 %, and modulus of 183 g/den, close to that of native spider silk (breaking tenacity of 10.6 g/den) [10Xia]. It was reported that the properties of the fibers were related to the Mw and that proteins with lower Mw did not produce fibers with good properties.

In a similar study, large spider egg case silk was engineered with repetitive and terminal domains found in spider silk and expressed in E. coli [13Lin]. Protein yields of up to 40 mg/l and purity of about 90 % were obtained. The proteins developed were lyophilized and dissolved in HFIP and extruded into a coagulation bath containing zinc chloride and iron chloride. Fibers formed were drawn up to

five times their original length. The tenacity of the regenerated fibers was 2.7 g/den, elongation was 10 %, and modulus was 81 g/den compared to the tenacity of 2.0 g/ den, 60 % elongation, and a Young’s modulus of 52 g/den for the native egg case silk fibers [13Lin]. It was claimed that it was the first time to obtain regenerated protein fibers with properties higher than that of the native fibers. It was suggested that the presence of the metals during coagulation could have contributed to the increase in tensile strength [13Lin]. Influence of various ratios of the amino acid sequence GPGXX on the extensibility of regenerated spider silk fibers was studied by Brooks et al. [08Bro]. Argiope aurantia spiders were used to reproduce GPGXX motifs in three different levels with 16, 12, and 8 repeat times and protein sizes of 63, 71, and 67 kDa, respectively, and expressed in E. coli. Proteins obtained were dissolved in HFIP in 10-12 % concentrations and extruded into an isopropanol coagulation bath, and fibers were collected without drawing. Table 50.5 provides a comparison of the properties of the fibers obtained. As seen in Table 50.5, the fibers obtained have considerable differences in strength and elongation especially for the proteins containing eight repeat motifs. It was found that a corresponding increase in type II p-turns with increasing number of repeats and subsequent increase in extensibility was observed. It was also suggested that the molar percent of alanine did not have a direct correlation with strength, and the relationship between amino acid sequence and mechanical properties was not fully understood [08Bro].

The use of harsh solvents or inability to dissolve higher amounts of proteins is one of the primary reasons for the limited use of spider silk in commercial applications [14Ris]. Typically, ionic solvents are used to dissolve spider silk proteins as seen from the above discussions. However, it has been shown that spider silk proteins were dissolved in aqueous buffers and produced into fibers when the recombinant proteins were recovered from conditioned culture media [02Laz]. Spider silk proteins were expressed in bovine mammary epithelial alveolar cells and in baby hamster kidney cells [02Laz]. Proteins obtained had molecular weights between 110 and 140 kDa. Solution concentrations up to 250 mg/ml were obtained depending on the type and size of protein (42-55 kDa) used [02Arc]. Fibers were of 10-60 pm in diameter and had toughness and modulus in comparison to those of native dragline silk fibers. It was reported that protein concentrations above 23 % were necessary to form fibers and the coagulation bath consisted of at least 20 % water and drawn in methanol up to 70-80 %. Except for lower tenacity, the toughness, modulus, and elongation of the regenerated fibers were higher than that of the native dragline silk fibers [02Laz].

Dragline silk was harvested from female N. clavipes spiders to yield about 0.8 mg of dragline silk per spider per day [98Sei]. The silk obtained was later dissolved in hexafluoro-2-propanol (HFIP) in concentrations of 1 % (w/w) and extruded into acetone coagulation bath. Fibers obtained have an average diameter of 40 pm compared to a diameter of 2.5-4 pm in native silk fibers. Proteins in the regenerated fibers had minimum p-sheets, but immersion and drawing of the fibers in water lead to a threefold increase in p-sheet content leading to substantial increase in the tensile properties of the fibers [00Sei]. The NMR spectrum of the fibers was similar to that of the native silk fibroin. The highest strength obtained from the regenerated spider silk fibers was 2.8 g/den and modulus was 70 g/den, much lower compared to the strength of 7.6 g/den and modulus of 94.8 g/den for native spider silk fibers [00Sei].

A microfluidic device was used to assemble engineered and recombinantly produced spider silk proteins into fibers [08Ram]. Proteins from two variants (ADF3 and ADF4) of the dragline silk fibroin from Araneus diadematus were recombinantly produced using E. coli and referred to as eADF3 and eADF4. The ability of these two proteins to form fibers in vitro was studied. eADF3 readily forms spherical particles with diameters of 1.5 pm, but fibers were obtained when the phosphate concentration was 500 mM and pH was 6.0 [08Ram]. Highly aligned or coiled fibers as seen in Fig. 50.2 containing p-sheets were obtained by changing the flow in the microfluidic device. The addition of phosphate into eADF4 resulted in increased hydrophobic interactions resulting in dense packing of the proteins and a high density of p-sheets. No fibers were obtained for eADF4 as irreversible spheres were formed in the microfluidic device [08Ram]. Since natural spider silk proteins contain both eADF3 and eADF4, the ability of the mixture of these two proteins to form fibers was also studied. It was found that eADF4 was homogenously distributed in the fibers as seen in Fig. 50.2. It was concluded that formation of colloidal aggregates was necessary for fiber formation under the conditions studied. These two protein variants were reported to determine the solubility of the proteins [04Hue]. In this approach of using a mixture of two proteins, a concentrated protein solution was pulled through a spinning duct, and the pH was decreased from slightly basic to slightly acidic, and phosphate and potassium ions were added to salt out the proteins. Composition of the individual and combined proteins is shown in Table 50.6. A microfluidic device was also used to fabricate fibers using small quantities (50 pl) of regenerated protein solution obtained from B. mori [11Kin]. Fibers obtained from the microfluidic device had diameters of 20-45 pm and strength of about 0.6 g/den much lower than that of native B. mori silk.

A chimeric silk-like protein encoding the synthetic gene that resembled the polyalanine-encoding region in Samia cynthia ricini silk and another sequence from B. mori silk was regenerated in E. coli. Proteins obtained had predominant a-helix structure and improved solubility and could be dissolved in 8 M urea [03Asa]. However, the ability of the recombinant proteins to form fibers and the structure and properties of the fibers were not studied.

Recombinant proteins have been used to understand the reasons for the high extensibility but lower strength of flagelliform silk compared to dragline silk

[13Adr]. Four distinct motifs (Table 50.7) found in flagelliform silk were constructed and inserted into a plasmid vector and then cloned into E. coli strain BL21DE3. Proteins were expressed, harvested, and purified and used for fiber production by dissolving 15 % proteins in HFIP and extruded into a 100 % isopropyl alcohol bath. Fibers were later drawn up to three times to align the molecules. Properties of the fibers obtained before and after drawing are displayed in Table 50.8. Tensile properties of the fibers listed in Table 50.8 show that the GY

and G fibers have low strength, whereas the GF and GFY fibers were considerably stronger suggesting that the spacer (F) was responsible for the strength. Similarly, the GGC motif provided substantially higher elongation to the fibers. It was suggested that the spacer promotes or stabilizes the secondary structure between two adjacent modules leading to stronger fibers. The strongest fibers obtained were from the GFY motif which is also found in the native Flag silk. It was concluded that the GGX motif contributed to extensibility and that the spacer contributes to the strength of the fibers.

A novel hybrid fiber that combined the polyalanine region of the Samia cynthia ricini silk and the cell adhesion region (RGD) derived from fibronectin was developed by Asakura et al. [04Asa]. In their study, the two proteins were expressed in E. coli, and the proteins obtained were dissolved in formic acid or trifluoroacetic acid. Although fibers were not produced, films were made and used to determine the cell adhesion and growth using African green monkey kidney cells. It was reported that the conformation of the polyalanine region could be controlled and that higher cell adhesion and growth were seen on the hybrid proteins compared to collagen.

Recombinantly produced silk-elastin-like proteins containing repeat units from silk and elastin were made into fibers through wet spinning [09Qiu]. The addition of the silk-like elastin proteins was reported to impart stimulus sensitivity that could provide temperature — and pH-sensitive hydrogels. Proteins were dissolved in formic acid and wet spun into a methanol/water coagulation bath. Fibers were later cross — linked with glutaraldehyde vapors to improve tensile properties and water stability. Fibers with length up to several meters were obtained using a protein concentration of 25 % and had strength between 0.02 and 0.7 g/den but low elongation of 2 % and modulus between 8.7 and 43.5 g/den. About 50-60 % p-strands in the form of P-tum conformation and p-strand structures were detected. When wet, the fibers had considerably poor strength and modulus, but the elongation increased to more than 700 %. However, cross-linking with glutaraldehyde increased the wet stability and provided fibers with poor wet strength of about 0.2 g/den and high elongation between 200 and 700 %.

References

[97Fah1] Fahnestock, S. R., Irwin, S. L.: Appl. Microbiol. Biotechnol. 47, 23 (1997)

[97Fah2] Fahnestock, S. R., Bedzyk, L. A.: Appl. Microbiol. Biotechnol. 47, 33 (1997)

[98Sei] Seidel, A., Liivak, O., Jelinski, L. W.: Biomacromolecules 31, 6733 (1998)

[00Sei] Seidel, A., Liivak, O., Calve, S., Adaska, J., Ji, G., Yang, Z., Grubb, D., Zax, D. B.,

Jelinski, L. W.: Macromolecules 33, 775 (2000)

[01Sch] Scheller, J., Guhrs, K., Grosse, F., Conrad, U.: Nat. Biotechnol. 19, 573 (2001)

[02Arc] Arcidiacono, S., Mello, C. M., Butler, M., Welsh, E., Soares, J. W., Allen, A., Zeigler,

D., Laue, T., Chase, S.: Macromolecules 35, 1262 (2002)

[02Laz] Lazaris, A., Arcidiacono, S., Huang, Y., Zhou, J., Duguay, F., Chretien, N., Welsh, E.

A., Soares, J. W., Karatzas, C. N.: Science 295, 472 (2002)

[02Won] Wong, C., Kaplan, D. L.: Adv. Drug Deliv. Rev. 54(8), 1131 (2002)

[03Asa] Asakura, T., Nitta, K., Yang, M., Yao, J., Nakazawa, Y., Kaplan, D. L.: Biomacro­

molecules 4, 815 (2003)

[04Asa] Asakura, T., Tanaka, C., Yang, M., Yao, J., Kurokawa, M.: Biomaterials 25, 617 (2004)

[04Hue] Huemmerich, D., Scheibel, T., Vollrath, F., Cohen, S., Gat, U.: Curr. Biol. 14, 2070 (2004)

[05Mot] Motohashi, T., Shimojima, T., Fukagawa, T., Maenaka, K., Park, E. Y.: Biochem.

Biophys. Res. Comm. 326, 564 (2005)

[07Ven] Vendrely, C., Scheibel, T.: Macromol. Biosci. 7, 401 (2007)

[08Bro] Brooks, A. E., Stricker, S. M., Joshi, S. B., Kamerzell, T. J., Middaugh, C. R., Lewis, R. V.: Biomacromolecules 9, 1506 (2008)

[08Ram] Rammensee, S., Slotta, U., Scheibel, T., Bausch, A. R.: Proc. Natl. Acad. Sci. USA 105(18), 6590 (2008)

[09Qiu] Qiu, W., Teng, W., Cappello, J., Wi, X.: Biomacromolecules 10, 602 (2009)

[10Xia] Xia, X., Qian, Z., Ki, C. S., Park, Y. W., Kaplan, D. L., Lee, S. Y.: PNAS 107(32),

14059 (2010)

[11Kin] Kinahan, M. E., Filippidi, E., Koster, S., Hu, X., Evans, H. M., Pfohl, T., Kaplan, D. L., Wong, J.: Biomacromolecules 12, 1504 (2011)

[12Chu] Chung, H., Kin, T., Lee, S. Y.: Curr. Opin. Biotechnol. 23, 957 (2012)

[12Teu1] Teule, F., Addison, B., Cooper, A. R., Ayon, J., Henning, R. W., Benmore, C. J.,

Holland, G. P., Yarger, J. L., Lewis, R. V.: Biopolymers 97, 418 (2012)

[12Teu2] Teule, F., Miao, Y., Sohn, B., Kim, Y., Hull, J. J., Fraser, M. J., Lewis, R. V., Jarvis, D.

L.: Proc. Natl. Acad. Sci. USA 109(3), 923 (2012)

[13Adr] Adrianos, S. L., Teule, F., Hinman, M. B., Jones, J. A., Weber, W. S., Yarger, J. L., Lewis, R. V.: Biomacromolecules 14, 1751 (2013)

[13Lin] Lin, Z., Deng, Q., Liu, X., Yang, D.: Adv. Mater. 25, 1216 (2013)

[13Tok] Tokareva, O., Michalcxechen-Lacerda, V. A., Rech, E. L., Kaplan, D. L.: Microb.

Biotechnol. 6(6), 651 (2013)

[14Ris] Rising, A.: Acta Biomater. 10(4), 1627 (2014)

[14Wei] Weichert, N., Hauptmann, V., Menzel, M., Schallau, K., Gunkel, P., Hertel, T. C., Petzch, M., Spohn, U., Conrad, U.: Plant Biotechnol. J. 12(2), 265 (2014)

Biothermoplastics from Renewable Resources

Keywords

Starch • Lactide • Fermentation • Extrusion • Dyeability • Hydrolysis • Crystallinity

Poly(lactic acid) commonly known as PLA is produced by condensation polymeri­zation from lactic acid which is derived by fermentation of sugars from carbohy­drate sources including corn and sugar cane. Commercial production of PLA is through the conversion of lactide to PLA through ring opening polymerization catalyzed by a Sn(II)-based catalyst [10Gro]. The processing, properties, and potential applications of PLA are mainly dependent on the ratio of the L — and D — isomers of lactic acid. Among the different forms of PLA that can be derived, stereo-complex type polylactides that consist of both enantiomeric poly(lactic acid) and poly(D-lactic acid) are high performance polymers with melting temperature of 230 °C, higher (50 °C) than that of PLLA or PDLA. Some of the properties of the stereo-complex PLA and PLLA are provided in Table 66.1 in comparison to poly (glycolic acid) (PGA) and poly(3-hydroxybutyrate) (PHB) [10Hir]. PLA and its isomers have been blended with various other synthetic and natural biopolymers to produce blends. PLA can be solution spun or melt spun into fibers, but generally, the latter is more economical and environmentally friendly and also produces fibers with better properties [10Aga]. However, melt spinning of PLA can cause signifi­cant hydrolytic degradation and, therefore, solution spinning of PLA is used to obtain fibers with high performance properties. Some of the fiber production conditions and the properties of the fibers obtained are listed in Table 66.2.

A novel method of reactive extrusion was used to produce PLA fibers through ring opening polymerization using a new catalytic system [01Sch]. Commercially available PLA (LA 0200 K) composed of L-lactic acid (92 %) and meso lactic acid (8 %) was extruded at 170, 180, and 185 °C and drawn to about 4-6 draw ratios at temperature of 65 °C in the first zone and 110 °C in the second zone. Take-up speeds of up to 5,000 m/min were used and the effect of spinning speed and draw

ratios on the properties of the fibers were studied. Table 66.3 provides a comparison of the properties at the different fiber production conditions.

The relatively low melting temperature (160-180 °C) of PLA poses considerable problems in processing the fibers for various applications. One of the simplest approaches to increase the melting temperature of PLA by almost 50 °C is to produce a stereo-complex crystal by blending equal ratios of PLLA and poly(D — lactic acid). Furuhashi et al. used this method and developed stereo-complex fibers and studied the properties of the fibers after various processing conditions [06Fur]. Draw ratio and annealing temperature were found to determine the extent of homo — and stereo-complex crystals formed. As seen in Fig. 66.1, X-ray diffrac­tion patterns of fibers drawn at 120 °C and annealed between 120 and 170 ° C showed reflections from both the homo — and stereo-complex. However, fibers annealed at 180 and 190 °C had reflections from the stereo-complex alone. Highest strength of the fibers obtained was 2.7 g/den and modulus was 38 g/den, signifi­cantly higher than previous reports on producing PLA fibers with high stereo­complex content.

Substantial changes in the structure and properties of the PLA fibers were observed with change in the take-up velocities. It was proposed by optimizing the take-up velocities; the cold drawing of the fibers could be avoided [14Zak]. Diameter of the fibers decreased linearly (38 to 11 qm) when the take — up velocity was increased from 100 to 400 m/min. Changes in diameter of the fibers were found to be associated with the changes in the orientation of the fibers. Based on dichroic ratio (1/D value) of the C=O stretching band at 1,751 cm-1, it was found that fibers with larger diameters had poor orientation along the transverse direction of the fiber whereas the thinner fiber had much better orientation. A schematic representation of the changes in the orientation of the fibers with diame­ter of the fibers is shown in Fig. 66.2 [14Zak]. Consequent to the increasing orientation, the crystallinity (%) of the fibers also increased from about 15 to 35 %. Tensile properties of the fibers were also found to increase substantially with decreasing diameters [14Zak].

PLA has been blended with other biopolymers to improve the ductility and crystallization. Poly(butylene succinate) (PBS) was blended with PLA in a single screw extruder and extruded at 190-210 °C as filaments [13Jom]. Some of the

Table 66.2 Influence of fiber forming conditions on the properties of the fibers [lOHir] Initial Mw (x 1СГ3) Extrusion temp (°С) Collection speed (m/min) Nozzle diameter (mm) As-spun fiber crystallinity (%) Final Mw (x 10~3) Drawn fiber diameter (pm) Fiber strength (g/den) Fiber modulus (g/den) 19-182 160-190 — 0.13-3.8 — <114 25-500 3.8-5.5 56 <300 185 0.25-0.35 1 — 180-260 — 4.0 56 360 200 — 1 5 110 150 5.6 68 98 190 — 0.5 — 38 76 3.2 — 280 210 1 0.25 42 100 83 4.2 72 330 240 2 (5-20) 1 5 (20—40) 110 80 7.0 72 — — 2,000-5,000 — — — — 3.1 48 207 185 1,000-5,000 0.3 0-30 180 — 2.4 32

Intensity of C=0 stretching vibration increases at the transverse direction with the chain orientation

Fig. 66.2 Schematic representation of the changes in the orientation of the fibers with decreasing fiber diameters [14Zak]. Reproduced with permission from Elsevier

properties of the PLA and PBS in the blends are listed in Table 66.4. As seen in the table, there are two distinct melting temperatures between 169-172 °C and ПО- 113 °C which are the melting temperature of PLA and PBS, respectively. Tensile property evaluation showed that addition of 10 % of PBS into PLA increased the tensile strength and modulus but decreased substantially at higher levels of PBS.

SEM image of the fracture surface of the blends showed distinct phase separated regions as seen in Fig. 66.3. Based on the distinct melting temperatures recorded and phase separation seen in the SEM pictures, it was concluded that PBS and PLA were not compatible. It was also suggested that presence of PBS up to 10 % in the blend acted as nucleation sites for PLA and improved the crystallinity [13Jom].

Dyeing of PLA, especially in blends with cotton, is considered to be a challenge due to the degradation of PLA in alkaline conditions. The effects of dyeing conditions on the thermal behavior of PLA fibers were investigated by Bilal et al. [11Bil]. Four different disperse dyes with long alkyl chains were used to dye the fibers and the changes in the polymer structure was studied using differen­tial scanning calorimetry (DSC) and X-ray diffraction. Increasing temperature 70 °C was found to increase dye uptake and crystallinity increased from 53 to 60 % when the fibers were dyed at 130 °C [11Bil]. As temperature increased, the fiber was able to swell and increase its chemical potential leading to better dye sorption. Crystallinity of the fibers increased from 50 to 60% but no changes were observed in the crystal structure. Cold crystallization temperature and melting enthalpy varied with changing temperature as seen in Fig. 66.4. Dye exhaustion (%) increased continually with increase in pH for all the dyes as seen in Fig. 66.5.

After dyeing of PLA fibers with disperse dyes, dye molecules are deposited on the surface of the fibers due to their insolubility in water. A reductive clearing process is used to remove the surface deposited dye and increase fastness properties [11Avi1]. Changes in the color fastness, degradation, and molecular weight were studied using 11 different commercially available clearing agents. The extent of color fastness after the clearing treatment was dependent on the dyeing conditions used. As seen in Table 66.5, substantial improvement in color fastness was obtained after clearing but the extent of improvement was different for the different dyes and dyeing conditions used [11Avi1]. Changes in the molecular weight of the fibers

after dyeing are displayed in the table (Table 66.5). Molecular weight of the fibers decreased to 48.2 from 61.1 suggesting polymer degradation during dyeing. Over­all, clearing resulted in a slight decrease in color yield, higher lightness, and brighter appearance without significant shade change [11Avi1]. In further continu­ation of their work, Avinc discovered that lower air content in the sealed reduction­clearing equipment and higher amounts of sodium dithionite (reducing agent) provided better wash fastness values. Similarly, reduction clearing with higher liquor ratio reduced the amount of air and therefore provided higher wash fastness values. The treatments adopted did not cause polymer degradation [11Avi2].

PLA fibers were treated with enzymes to improve dyeability and handling [07Saw]. PLA fabrics that were woven and later desized were used for the study.

Fabrics were treated in the enzyme solution for one week at 35 °C and fabric to enzyme solution ratio of 1:50. Enzyme concentration in the solution was 2 g/L and pH was 7. After the desired treatment time, the fabrics were weighed to determine the weight loss and tensile tested to know the reduction in tensile properties [07Saw]. As seen in Fig. 66.6, the PLA fabrics have strength loss varying from 5 to 25 % depending on the type of enzyme used. Similarly, the PLA fibers were found to have higher weight loss than wool fibers treated with the enzymes under the same conditions.

In a similar study, PLA fabrics were treated with two lipolytic enzymes, lipase and esterase, at pH 8.0, temperature of 40 °C, and an enzyme concentration of 10 % on weight of the fabric as the optimum conditions [13Lee]. The treated fabrics had a weight loss of only 0.7% without change in the tensile strength. However, the moisture regain of the samples nearly doubled and a new crystalline peak attributed to the morphological changes in the fibers was observed. The degree of crystallinity of the untreated fabrics was 42 % and that of the esterase treated fabrics increased to 67 % [13Lee].

Apart from the enzyme studies, PLA has also been ozonated to make it more suitable for textile applications [11Ere]. Ozonated fabrics had about 6 % increase in whiteness after treating in ozone for 10 min, time of wetting decreasing by about 20 %, and flexural rigidity by about 16 %. Fabric strength did not decrease after treating for 10 min but about 10 % decrease was seen after ozonating for 60 min. It was suggested that ozone treatment could be a more convenient and safe treatment than peroxide treatment of PLA fabrics [11Ere].

References

[01Sch] Schmack, G., Jehnichen, D., Vogel, R., Tandler, B., Beyreuther, R., Jacobsen, S., Fritz, H. G.: J. Biotechnol. 86, 151 (2001)

[06Fur] Furuhashi, Y., Kimura, Y., Yoshie, N., Yamane, H.: Polymer 47, 5965 (2006) [07Saw] Sawada, K., Urakawa, H., Ueda, M.: Text. Res. J. 77, 901 (2007)

[10Aga] Agarwal, A. K.: In: Auras, R., Lim, L., Selke, S. E.M., Tsuji, H. (eds.) Poly(lactic acid): Synthesis, Structures, Properties, Processing and Applications. Wiley, Danvers (2010) [10Gro] Groot, W., Krieken, J. W., Sliekersl, O., Vas, S. D.: In: Auras, R., Lim, L., Selke, S. E.

M., Tsuji, H. (eds.) Poly(lactic acid): Synthesis, Structures, Properties, Processing and Applications. Wiley, Danvers (2010)

[10Hir] Hirata, M., Kimura, Y.: In: Auras, R., Lim, L., Selke, S. E.M., Tsuji, H. (eds.) Poly (lactic acid): Synthesis, Structures, Properties, Processing and Applications. Wiley, Danvers (2010)

[11Avi1] Avinc, O.: Text. Res. J. 81, 1049 (2011)

[11Avi2] Avinc, O.: Text. Res. J. 81, 1158 (2011)

[11Bil] Bilal, M. B., Viallier-Raynard, P., Haidar, B., Colombe, G., Lallam, A.: Text. Res. J. 81, 838 (2011)

[11Ere] Eren, H. A., Avinc, O., Uysal, P., Wilding, M.: Text. Res. J. 81, 1091 (2011)

[13Jom] Jompang, L., Thumsorn, S., Wong, J., Surin, P., Apawet, C., Chaialermwong, T., Kaabbuathong, N., O-Charoena, N., Srisawate, N.: Energy Procedia 34, 493 (2013) [13Lee] Lee, S. H., Song, W. S.: Text. Res. J. 83, 229 (2013)

[14Zak] Zakir Hossain, K. Z., Parsons, A. J., Rudd, C. D., Ahmed, I., Thielemans, W.: Eur. Polym. J. 53, 270 (2014)

Natural Cellulose Fibers from Renewable Resources

Keywords

Borassus flabellifer • Husk • Empty fruit bunch fiber • Lignocellulose

Palm trees are grown for oil in about 15 million hectares, and about 11 tons of dry mass are produced per hectare of palm grown. Cultivation of palm trees generates by-products called fronds (leaves) shown in Fig. 6.1, and about 164 million tons of fronds are estimated to be produced every year in the world [00Dah, 00Lin, 08Kha]. In addition to the fronds, the palm plants provide two additional sources of fibers. After harvesting the seeds, the fibrous empty fruit bunches (oil palm empty fruit bunch) (OPEFB) have been studied as potential sources for fibers. A kilogram of fruit bunch produces approximately 22 g of palm oil but results in about a kilogram of OPEFB [09Gun, 13Kit]. Similarly, the mesocarp left in the seed after squeezing for oil is also considered a source for fibers. On an average, about 400 g of fibers can be obtained from each OPEFB by natural retting [97Sre]. OPEFB fibers typically are composed of about 63 % cellulose, 18 % hemicellulose, and 18 % lignin. Natural cellulose fibers have been extracted from various types and parts of the palm tree. Native to upper Africa, Hyphaene thebaica (doum palm) was used as a source to extract fibers from the folioles and leaf stalks. For mechanical extraction, the plant parts were separated into fibers by beating and grating to liberate the fibers that were later dried in air [09Sgh]. After mechanical extraction, fibers were further treated with NaOH (3 N) for 2 h at 90 °C and later with sodium hypochlorite at room temperature [09Sgh]. As seen in Table 6.1, considerable variations in fiber properties can be seen between the foliole fibers and the leaf stalk fibers. Similarly, alkali treatment resulted in fibers with considerably finer diameter and higher strength, elongation, and modulus for the fibers obtained from the folioles. Morphologically, the fibers contain pores on the surface that were more evident after alkali treatment as seen in Fig. 6.2 [09Sgh].

Fibers obtained from empty fruit bunches have better tensile properties than the fibers obtained from the folioles and leaf stalks of doum palm reported above. The

diameter of the fibers was also reported to have a significant effect on the tensile properties [09Gun]. For instance, fibers with diameters between 400 and 475 qm had tensile strength of 1.9 g/den compared to 0.7 g/den for fibers with diameters between 575 and 720 qm [09Gun]. OPEFB fibers have been treated with chemicals to reduce their hydrophilicity and improve their compatibility with synthetic polymers. Figure 6.3 shows the surface features of the untreated, sodium hydroxide-treated, and succinic anhydride-treated OPEFB fibers. Chemical treat­ment results in the removal of the cavities and the surface became smoother and the silica deposits (circled) could be clearly seen [07Law]. Such modifications also result in considerable changes in tensile properties as seen in Table 6.2.

Borassus flabellifer L. (palmyra palm) trees that grow in tropical areas have been used to obtain fibers from the leaves and also from the husk of the fruits. These trees belong to the Arecaceae family, grow to about 25-30 m in length, have life spans of about 100 years, and produce fruits 4-7 in. in diameter [87Dav]. Mostly processed by hands, the leaves and husks are treated to obtain fibers that are made into various types of products. In its simplest form, the leaves are cut into strands of required dimensions and then woven into mats or baskets. Leaves at the base of the plants

collected from trees that are 5-10 years old are beaten with a mallet to remove the outer covering and obtain fibers. Fibers as long as 45 cm with good strength and elongation are obtained and are mostly used as bristles in brushes and also as cordage. Unfortunately, there is not much information available on the properties of the fibers obtained from the leaves of the Borassus fibers. In addition to the leaves, the husks of the fruits of Borassus flabellifer can also be used to produce fibers.

As seen in Fig. 6.4, an outer black shell forms the husk with the fibers that are separated to get the fruits inside. Coarse and fine fibers are separated from the husks mechanically followed by beating and are brown in color as seen in Fig. 6.5. Fibers obtained from the husks after extracting with ethanol and carbon tetrachloride had diameters of about 120 ^m, length of about 81 mm, and linear density of 43 tex [08Sar]. Interestingly, the fibers had similar dry and wet strength, whereas natural cellulose fibers from agricultural by-products such as cornhusks lose considerable strength when wet. Another study treated water-extracted Borassus fibers with three levels of NaOH at room temperature and studied the properties of the fibers.

Similarly, Borassus fruits were treated with alkali of various concentrations and different times, and it was observed that fine fibers with different tensile properties and chemical composition as given in Tables 6.3 and 6.4 could be extracted [13Red]. Although similar to coir fibers from the husks of coconuts, Borassus fibers are reported to have considerably lower amount of lignin (5.4 %) in one study, but lignin contents as high as 25 % have also been reported by others [12Boo, 13Red]. Another unique feature of the Borassus fibers is their considerably high

Fig. 6.4 Digital image of Borassus fruit reveals the thick outer shell (black) which is the source of fibers. Reproduced with permission from John Wiley and Sons [13Red]

elongation ranging from 30 to almost 50 % as seen in Table 6.3. Such high elongation is rarely seen in natural cellulose fibers and is mainly due to the low microfibrillar angle and poor orientation of the cellulose crystals along the fiber axis. Despite the high elongation, the fibers seem to have high modulus of 85 g/den before alkali treatment and increased to 269 g/den after treating in alkali [13Red]. Morphologically, Borassus fibers have hollow centers and appear like tubes as seen in Fig. 6.6 that could help in moisture and sound absorptions. Alkali treatment results in the destruction of these tubes, and fibrillation could be observed on the surfaces [09Red].

Similar to Borassus flabellifer, fibers have also been produced from sugar palm (Arenga pinnata) [12Ish]. In Malaysia, fibers, traditionally called ijuk fibers

NJ О

Table 6.3 Influence of alkali treatment time on the tensile properties and chemical composition of Borassus fruit fibers Treatment time [h] Diameter [mm] Tensile properties Chemical composition Strength [g/den] Elongation [%] Modulus [g/den] Cellulose [%] Hemicellulose [%] Lignin [%] 0 0.140 0.5 ±0.03 35 ±4 85 ±38 53 ±1.2 30± 1.4 17 ±1.5 1 0.135 0.80 ±0.06 43 ±4 200 ±46 60 ±1.1 16± 1.2 23 ±1.3 4 0.127 0.91 ±0.04 51 ±5 254 ±62 62 ±1.3 14 ± 1.2 24± 1.1 8 0.118 0.93 ± 0.05 58 ± 6 269 ± 38 63 ±1.2 13 ± 1.2 24 ±1.4 12 0.112 0.78 ±0.03 36±3 200 ±31 62 ±1.1 12 ± 1.2 25 ±1.2 From [13Red]

o

Table 6.4 Influence of alkali concentration on the tensile properties and chemical composition of Borassus fruit fibers Tensile properties Chemical composition Alkali concentration Diameter [mm] Strength [g/den] Elongation [%] Modulus [g/den] Cellulose [%] Hemicellulose [%] Lignin [%] 0 0.140 0.5 ± 0.03 35 ±4 65 ±29 53 ±1.2 30± 1.4 17 ±1.5 2 0.135 0.6 ±.08 42 ±1 96 ±20 57 ± 0.7 24 ±1.2 19 ±1.1 5 0.123 0.7 ± 0.06 41 ±0.4 143 ± 10 60 ±1.4 17 ±0.7 22 ±0.5 10 0.112 0.7 ± 0.02 53 ±1.2 166 ± 30 62 ±1.0 13 ±0.5 25 ±0.8 15 0.099 0.8 ±0.04 42 ±1.0 198 ±50 64 ±0.4 11 ± 1.3 26± 1.8 20 0.090 0.4 ±0.05 45 ±3.2 155 ±10 62 ± 0.2 9.7 ± 1.7 28 ± 1.6 From Reddy [12Red]

obtained from sugar palm trees, have been used and sold for several centuries and mainly used as cordage to anchor ships due to the relatively high resistance of the sugar palm fibers to seawater [12Ish]. It has been reported that the parts of the sugar palm tree are naturally in fibrous form and can be directly used to prepare cordage without the need for chemical or mechanical extraction. Fibers were extracted from various lengths of the palm tree and analyzed for their structure and properties. Table 6.5 shows the tensile properties of the fibers obtained at various lengths of the palm trees. The fibers have properties similar to that of coir fibers and are distin­guished by their high elongation. However, the modulus of the fibers is unusually low compared to other lignocellulosic fibers. The highest strength and toughness of the fibers obtained were between 9 and 13m due to the degradation of the fibers at the bottom portions and immature and green fibers present at the top. In addition, the composition of the fibers also varied considerably with height as seen in Table 6.6. Figure 6.7 shows the cross section of the undegraded (left) and degraded (right) sugar palm fiber bundles. In terms of composition, the sugar palm fibers are similar to that of coir fibers with lignin content as high as 25 %.

Sugar palm stems were immersed in water to remove foreign materials, and the fibers obtained were tested for single fiber strength [10Bac]. The fibers had a tensile

strength of 1.5 g/den, elongation of 19.6 %, and a low modulus of 28 g/den. Morphologically, the fibers had a rough surface and perforations which were supposed to promote adhesion to the matrix.

References

[87Dav] Davis, T. A., Johnson, D. V.: Econ. Bot. 41(2), 247 (1987)

[97Sre] Sreekala, M. S., Kumaran, M. G., Thomas, S.: J. Appl. Polym. Sci. 66(5), 821 (1997) [00Dah] Dahlan, I.: Asian Australas. J. Anim. Sci. 13, 300 (2000)

[00Lin] Lin, K. O., Zainal, Z. A., Quadir, G. A., Abdullah, M. Z.: Int. Energy J. 1(2), 77 (2000) [03Sre] Sreekala, M. S., Thomas, S.: Compos. Sci. Technol. 63, 861 (2003)

[07Law] Law, K., Daud, W. R.W., Ghazali, A.: Bioresources 2(3), 351 (2007)

[08Kha] Khalil, H. P.S. A., Alwani, M. S., Ridzuan, R., Kamarudin, H., Khairul, A.: Polym. Plast. Technol. Eng. 47, 273 (2008)

[08Sar] Saravanan, D., Pallavi, N., Balaji, R., Parthiban, R.: J. Text. Inst. 99(2), 133-140 (2008)

[09Gun] Gunawan, F. E., Homma, H., Brodjonegoro, S. S., Baseri Hudin, A. B., Zainuddin, A. B.: J. Solid Mech. Mater. Eng. 3(7), 943 (2009)

[09Red] Reddy, K. O., Guduri, B. R., Varadarajulu, A.: J. Appl. Polym. Sci. 114, 603 (2009) [09Sgh] Sghaier, S., Zbidi, F., Zidi, M.: Text. Res. J. 79(12), 1108 (2009)

[10Bac] Bachtiar, D., Sapuan, S. M., Zainudin, E. S., Khalina, A., Dahlan, K. Z.M.: Mater. Sci. Eng. 11, 1 (2010)

[11Bha] Bhat, I., Abdul Khalil, H. P.S., Ismail, H., Alshammari, T.: Bioresources 6(4), 4673 (2011)

[12Boo] Boopathi, L., Sampath, P. S., Mylsamy, K.: Compos. Part B 43, 3044 (2012)

[12Ish] Ishak, M. R., Sapuan, S. M., Leman, Z., Rahman, M. Z.A., Anwar, U. M.K.: J. Therm.

Anal. Calorim. 109, 981 (2012)

[12Red] Reddy, K. O., Shukla, M., Maheshwari, U. C., Varada Rajulu, A.: J. For. Res. 23(4), 667 (2012)

[13Kit] Kittikorn, T., Stromberg, E., Ek, M., Karlsson, S.: Bioresources 8(2), 2998 (2013) [13Red] Reddy, K. O., Maheshwari, C. U., Shukla, M., Song, J. I., Varadarajulu, A.: Compos. Part B. 44, 433 (2013)

Chitin, Chitosan, and Alginate Fibers

Keywords

Alginate • Chitosan • Blend fiber • Sorption • Antimicrobial properties • Biocompatibility • Drug loading • Drug release

Combining the inherent ability of alginate to form gels and sorb moisture with the antimicrobial activity of chitosan and other unique properties of the two polymers would provide ideal materials for various medical applications. Therefore, polyion complex fibers were prepared by combining the advantages of alginate and chitosan polymers for potential use as cartilage tissue engineering scaffolds. Since the anionic nature of alginate was not conducive for the attachment of chondrocytes, it was hypothesized that adding chitosan, a cationic polysaccharide with excellent cell adhesive properties, would improve chondrocyte adhesion [04Iwa]. Sodium alginate (6 %, Mw = 600,000) was mixed with chitosan (0.035 and 0.05 % on weight of alginate) and extruded into a CaCl2 bath to form fibers. The influence of the addition of chitosan on the tensile properties of the alginate fibers is shown in Table 29.1. Although the addition of chitosan did not change the tensile properties of the fibers, it was found that the composite fibers had enhanced cell attachment and proliferation, in vitro [04Iwa]. SEM image of chondrocytes shown in Fig. 29.1 on the alginate fibers containing 0.05 % chitosan showed the characteristic round morphology and dense collagen II fiber formation indicating good biocompatibility.

In a similar approach, chitosan (0.05 or 1 %) was added into alginate fibers, and the hybrid fibers were used as scaffolds for ligament and tendon tissue engineering [05Maj]. The hybrid fibers were found to have improved adhesion capacity for fibroblasts that produced type I collagen indicative of good biocompatibility.

In another study, alginate and chitosan were blended to form “alchite” fibers, and the antimicrobial activity of the blend fibers was studied [11Mir]. Three different types of alginates and two types of commercially available chitin were used to compare the antimicrobial activity of various bacteria. Figure 29.2 shows the zone

Fig. 29.1 SEM image of rabbit articular chondrocytes seeded on alginate-based 0.05 % chitosan hybrid fiber after 14 days of culture. From Iwasaki

et al. [04Iwa]. Reproduced with permission from the American Chemical Society

of inhibition (cm) for the various fibrous samples studied in that research. As seen from the figure, all the alchite fibers were more effective against Micrococcus luteus B 110.

Since chitin and chitosan are not soluble in water, chitosan was carboxymethylated, and the water-soluble carboxymethyl chitosan (CMC) was blended with alginate and made into fibers [06Fan] with the expectation of better compatibility between alginate and carboxymethylated chitosan. The blend fibers were also treated with silver nitrate and N-(2-Hydroxy)-propyl-3- triethylammonium chitosan chloride (HTCC) to improve the antibacterial activity of the fibers. No striations were seen on the surface of the fibers indicating good miscibility between chitosan and the alginate. Some of the properties of the alginate-CMC are given in Table 29.2. The addition of chitosan up to 30 % increased the strength and elongation of the fibers and also water retention. Treating the fibers with antimicrobial agents did not show any major influence on mechani­cal properties, but the antibacterial activity increased manifolds. As seen in

Table 29.2, fibers treated with silver nitrate had bacterial reduction (Staphylococcus aureus) higher than 99.99 %. However, the fibers had poor wet strength, and the antimicrobial activity against more common bacteria such as Escherichia coli was not studied.

Several researchers have also attempted to improve the mechanical properties and stability of alginate-chitosan blend fibers by adding various additives. In one such attempt, chitin nanowhiskers with an average length and width of 343 and 46 nm, respectively, were used to reinforce alginate, and the structure and properties of the fibers were studied. The addition of nanowhiskers at low levels (0.2 %) increased the tenacity, elongation, and thermal stability of the fibers due to intermolecular hydrogen bonding and electrostatic interactions between alginate and chitin. Tenacity of the fibers obtained was between 1.0 and 1.2 g/den, and elongation was between 20 and 30 %. The presence of the nanowhiskers increased the biodegradation of the fibers in the presence of lysozymes, whereas the presence of Ca2+ ions in Tris-HCl buffer improved the tenacity of the fibers [08Wat]. In a different study, the same research group had reported that adding 1 % of chitosan nanowhiskers increased the strength of the fibers from 0.7 to about 1.1 g/den, and elongation had decreased to about 25 % from the initial value of 48 % [10Wat]. Figure 29.3 shows an optical image of the fibers containing 0.6 % chitosan nanowhiskers at two different magnifications. The release of the nanowhiskers from the fibers into Tris-HCl buffer was studied at 37 °C. A dose-dependent release was observed with half-life of about 1 h for fibers containing 0.6 % nanowhiskers. Addition of the nanowhiskers (1 %) led to a 43 % decrease in S. aureus and 84 % reduction in E. coli suggesting that the blend fibers could be useful for wound dressing and other applications.

Unlike the above-discussed approaches of directly blending alginate and chitosan, chitosan was formed into emulsion and the chitosan-citrate complex was used to produce chitosan-spotted alginate fibers [09Wat]. The emulsion approach was used to avoid the formation of gels when the oppositely charged chitosan and alginate molecules interact. Up to 10 % chitosan complex was added into the fibers, and the influences of chitosan content and fiber formation condition on tensile properties were investigated. A simple schematic of the process is shown

O/w-p rimary
emulsion micelle

complex Alginate/chitosan Chitosan-spotted

suspension alginate fibers

in Fig. 29.4. The addition of chitosan led to the fibers becoming striated, and increasing the level of chitosan led to the formation of spots or round protrusions on the fibers which were due to the emulsified chitosan-citrate complex. Tenacity and elongation of the fibers increased with the addition of 0.5 % chitosan complex but decreased substantially at higher levels of the complex. Fiber tenacities obtained were still considerably lower (0.8-6.2 g/den), and elongation (10-25 %) was typical of alginate fibers produced by other methods. Chitosan-loaded fibers were also reported to load higher amounts of anionic drugs compared to the neat fibers. However, the fibers containing 4 and 10 % chitosan had poor stability and disintegrated when immersed in PBS solution for 24 h.

References

[04Iwa] Iwasaki, N., Yamane, S., Majima, T., Kasahara, Y., Minami, A., Harada, K., Nonako, S., Maekawa, N., Tamura, H., Tokura, S., Shiono, M., Monde, K., Nishimura, S.: Biomacromolecules 5, 828 (2004)

[05Maj] Majima, T., Funakosi, T., Iwasaki, N., Yamane, S., Harada, K., Nonaka, S., Minami, A., Nishimura, S.: J. Orthop. Sci. 10, 302 (2005)

[06Fan] Fan, L., Du, Y., Zhang, B., Yang, J., Zhou, J., Kennedy, J. F.: Carbohydr. Polym. 65, 447 (2006)

[08Wat] Watthanaphanit, A., Supaphol, P., Tamura, H., Tokura, S., Rujiravanit, R.: J. Appl. Polym. Sci. 110, 890 (2008)

[09Wat] Watthanaphanit, A., Supaphol, P., Furuike, T., Tokura, S., Tamura, H., Rujiravanit, R.: Biomacromolecules 10, 320 (2009)

[10Wat] Watthanaphanit, A., Supaphol, P., Tamura, H., Tokura, S., Rujiravanit, R.: Carbohydr. Polym. 79(3), 738 (2010)

[11Mir] Miraftab, M., Barnabas, J., Kennedy, J. F., Masood, R.: J. Ind. Text. 40(4), 345 (2011)

Chitosan has been blended with other carbohydrates, proteins, and synthetic polymers to develop matrices with specific properties. Some of the chitosan blends that have been developed are listed in Table 58.3. As seen in the table, chitosan has been blended predominantly with synthetic polymers such as poly(vinyl) alcohol (PVA) and PEO due to their easy solubility but also with natural polymers like silk. Although aqueous acetic acid systems have been the main solvents, organic solvents such as dimethyl formamide and chloroform have also been used to dissolve and obtain chitosan fibers [09Lee].

Blends of chitosan (20-190 kDa) and alginate (80-120 kDa) nanofibers were produced by co-electrospinning the individual solutions with the aid of PEO to improve viscosity and DMSO and Triton X to assist in dissolution of the polymer [13Hu]. Bicomponent fibers that had better biocompatibility to cells than the individual polymers were obtained. Degradation of the fibers could be controlled by varying the extent of cross-linking by treating with calcium chloride [13Hu].

Chitosan deacetylated with hexanoyl chloride was dissolved in chloroform and electrospun into fibers in the presence of pyridiniumformate to increase electrocon­ductivity. Some of the properties of the hexanoyl chitosan solution and the fibers obtained are shown in Table 58.4. As seen in the table, increasing concentration of chitosan increased viscosity and fiber diameters. Addition of pyridiniumformate substantially increased conductivity and reduced fiber diameters as seen from the table.

Chitosan was carboxyethylated to make it water soluble and later blended with PVA to produce electrospun scaffolds [08Zho]. Fibers with blend ratios of up to 50/50 chitosan/PVA and average diameters from 131 to 456 nm were obtained. However, the morphology of the fibers was heavily dependent on the blend ratio

Table 58.3 Blends of chitosan having various molecular weights and degree of acetylation with other polymers electrospun into fibers using different solvents Polymer blend Molecular weight (kDa) Degree of acetylation (%) Solvent Chitosan/PVA 120-1,600 82.5-90 Aqueous acetic acid Chitosan/PVA 120 82.5 Aqueous acrylic acid Chitosan/PEO 148, 276 82 Aqueous acetic acid Chitosan/UHMWPEO — >85 Aqueous acetic acid/DMSO Chitosan/PET — 85 Trifluoroacetic acid Chitosan/silk fibroin 220 86 Formic acid Chitosan/poly(lactic acid/caprolactone) 600 80 Aqueous acetic acid Chitosan/collagen 1,000 85 HFIP/trifluoroacetic acid Carboxymethyl chitosan/PVA 405 84.7 Water Carboxyethyl chitosan/PVA 405 Water Quatemized chitosan/poly(vinyl pyrrolidone) 380 80 Water Quatemized chitosan/PVA chitosan 400 80 Aqueous acetic acid Chitosan-PLA 600 85 Trifluoroacetic acid-methylene chloride Chitosan-PVA-PLA 165 90 Aqueous acetic acid Adopted from Lee et al. [09Lee]

and electrospinning conditions [08Zho]. Since both carboxymethylated chitosan (CMC) and PVA are water soluble, the scaffolds were cross-linked using glyceral — dehyde after which the fibers were found to be stable in water up to 48 h without change in morphology.

Miscellaneous Applications of Biofibers from Renewable Resource

Keywords

Biofiber • Renewable resource • Absorbent • Keratin • Chromium • Membrane filtration

Keratin biofibers separated from chicken feathers were used to prepare polyurethane-keratin membranes for the removal of hexavalent chromium [11Sau]. Table 75.1 shows the chromium removal efficiency of the polyurethane — keratin membranes. As seen from the table, up to 38 % removal could be achieved depending on the type of modification done for the keratin fibers.

Reference

[11Sau] Saucedo-Rivalcaoba, V., Martinez-Hemandez, A. L., Martinez-Barrera, G., Velasco — Santos, C., Rivera-Armenta, J. L., Castario, V. M.: Water Air. Soil Pollut. 218, 557 (2011)

Regenerated Cellulose Fibers

Keywords

Cellulose dissolution • Cellulose solvent • Alkali • Polymerization degree • Tenacity

A simple approach to producing regenerated cellulose fibers was to dissolve cellulose pulp using alkali. The principle and mechanism of dissolving cellulose in alkali solutions are depicted in Figs. 16.1 and 16.2. It has been proven that the solubility of cellulose in alkali solutions is mainly governed by the degree of breakdown of the intramolecular hydrogen bonding and also by the degree of polymerization [92Kam, 98Iso]. The presence of lignin was found to lower disso­lution, whereas the extent of hemicellulose did not affect the solubility [98Iso]. Several authors have used alkali solutions to produce regenerated cellulose films and fibers using cellulose from different sources [92Kam]. Alkali-soluble cellulose was prepared by exploding softwood pulp (DP of 331) with steam, and later, the pulp was dissolved in 9.1 % of NaOH precooled to 4 °C and used to extrude fibers. Fibers were produced with a fineness of 53-84 denier and had % crystallinity between 65 and 67 %. The tensile strength of the fibers varied from 1.5 to 1.8 g per denier, and the elongation was between 4.3 and 7.3 % depending on the conditions used during coagulation [92Yam]. Similar to the NaOH/urea system, the alkali system of dissolving cellulose was also limited by the degree of polymeriza­tion. Cellulose with relatively high DP (850) had limited solubility (26-37 %) in the alkali solutions [98Iso, 08Wan]. Contrarily, rayon which has a lower DP, poorly ordered crystalline region, and weak hydrogen bonding completely dissolved in alkali solutions [90Yok].

References

[90Yok] Yokoto, H., Sei, T., Horii, F., Kitamaru, R.: J. Appl. Polym. Sci. 41, 783 (1990)

[92Kam] Kamide, K., Okajima, K., Kowsaka, K.: Polym. J. 24(1), 71 (1992)

[92Yam] Yamashiki, T., Matsui, T., Kowsaka, K., Saitoh, M., Okajima, K., Kamide, K.: J. Appl. Polym. Sci. 44, 691 (1992)

[98Iso] Isogai, A., Atalla, R. H.: Cellulose 5, 309 (1998)

[08Wan] Wang, Y., Zhao, Y., Deng, Y.: Carbohydr. Polym. 72, 178 (2008)

Natural Protein Fibers

Keywords

Lacewing • Silk production • Lacewing life cycle • Humidity • Fiber properties • Structural transition

Lacewings are insects that lay their eggs on the tips of silken threads called egg stalks as seen from Fig. 40.1 [09Wei]. Unlike most silk-producing insects, green lacewing (Mallada signata, Neuroptera) produces two distinct types of silks depending on the life cycle of the insect [08Wei]. Silks produced by lacewing in the larval stage and during the final instar of cocoon production were found to be different. Primary structure of the lacewing silk is composed of motifs containing 16 amino acids with cysteine residues [12Bau]. The cocoon silk is composed of 49 kDa proteins, with >40 % alanine, and contains a-helical secondary structure, considerably smaller than the proteins (>200 kDa) seen in the classic p-sheet silks. In terms of secondary structure, lacewing silk was mainly composed of unique and distinct cross p-sheets that run perpendicular to the fiber axis unlike the silk produced by other insects. A model suggesting the arrangement of the cross

P-sheets in lacewing silk is shown in Fig. 40.2 [13Lin]. Atomic force measurements and calculations have shown that the lacewing silk has a bending modulus three times higher than that of silkworm fibers [09Wei]. Tensile properties of the silk were found to be highly dependent on the water content (relative humidity, RH) with modulus decreasing from 50 g/den to 11 g/den when the relative humidity was increased from 30 to 100 % and the corresponding change in breaking stress was from 2.0 to 0.6 g/den. This substantial change in properties due to change in humidity was supposed to be due to the transition of the cross p-sheets to parallel P-sheets caused by the weakening of the hydrogen bonds at high humidity [12Bau]. At low RH, the total strength of the hydrogen bonds in one layer of the stalk is higher than that of the disulfide bonds causing the fibers to absorb low energies. When the RH is high, the hydrogen bonds are weakened, and the disulfide bonds are now stronger than the sum of the hydrogen bonds in one layer causing the hydrogen bonds to break. Such breakage of the hydrogen bonds allows the rear­rangement of the p-sheets [12Bau]. SEM image (Fig. 40.3) showed thinning of the fibers after stretching which was not reversible, again indicating the transformation of the p-sheets. The simple process by which lacewing secretes silks is considered to be more suitable for producing recombinant proteins [12Bau].

References

[08Wei] Weisman, S., Trueman, H. E., Mudie, S. T., Church, J. S., Sutherland, T. D., Haritos, V. S.: Biomacromolecules 9, 3065 (2008)

[09Wei] Weisman, S., Okada, S., Mudie, S. T., Huson, M. G., Trueman, H. E., Sriskantha, A., Haritos, V. S., Sutherland, T. D.: J. Struct. Biol. 168(3), 467 (2009)

[12Bau] Bauer, F., Bertinetti, L., Masic, A., Scheibel, T.: Biomacromolecules 13, 3730 (2012) [13Lin] Lintz, E. S., Scheibel, T. R.: Adv. Funct. Mater. 23, 4467 (2013)

Bacterial cellulose is produced by various sources (Table 61.1) but predominantly from Gluconacetobacter xylinum via a four step enzymatic process that consists of (1) phosphorylation of glucose to glucokinase, (2) isomerization of glucose-6- phosphate to glucose-1-phosphate by phosphoglucomutase, (3) the synthesis of uridine diphosphate glucose from glucose-1-phosphate by UDP-glucose pyrophosphorylase, and (4) the synthesis of cellulose from UDP-glucose by cellu­lose synthase that essentially converts glucose into cellulose [13Ash, 13Sax]. In a typical bacterial cellulose, sub-elementary fibril assembles with adjoining fibrils into 20-50 nm wide flat and twisted ribbons. A scanning electron image of bacterial cellulose fibers is shown in Fig. 61.1. To further understand the structure, an atomic force investigation was used to analyze the mechanism of formation of bacterial cellulose from Gluconacetobacter xylinus [13Zha]. Bacteria was cultured in Hestrin-Schramm (HS) medium and incubated at 30 °C for 7 days and the growth of the bacteria at different stages of culture was observed. Fibril formation took at least 2 h and after 6 h of incubation, singe fiber bundles with average diameters of

8.7 ^m were developed and overlapping of two single fibers was also seen. Ribbons of fibers were observed after 16 h of incubation with average single fiber diameter of 15 ^m and height of 41 nm as seen from the AFM images in Fig. 61.2. Fibrils obtained were further purified with 0.1 N NaOH and SEM image in Fig. 61.3 shows the image of the fibers. Interestingly, twisting of the single fibers in a right-handed fashion was also seen. A schematic of the proposed fibril and ribbon formation has been depicted in Fig. 61.4.

Researchers have also suggested that the conditions during the growth of the cellulose also play a major influence on the properties of the cellulose obtained. For instance, Shi et al. demonstrated that changing the growth media influenced DP whereas shaking or static conditions did not show a significant difference. A continuous decrease in the DP from about 2,400 to about 2,150 from 3 to 5 days and then an increase up to 2,300 was seen when the culture time was increased from

9 to 15 days [13Shi]. Types of carbon source and addition of surfactants were also observed to affect the growth of cellulose. In addition, the formation of the BC pellicles was influenced by the condition of the culture, i. e., static or agitated conditions [13Ash]. As seen from Fig. 61.5 (left) and (right), static culture produced a continuous BC pellicle whereas agitated culture resulted in discontinuous pellicles and also frequently led to the formation of cellulose mutants and decline in cellulose synthesis [13Ash].

In addition to Gluconacetobacter xylinum, other sources of bacteria have also been used for cellulose production. The potential of producing bacterial cellulose using Gluconacetobacter intermedius CIs 26 and the properties of the fibers obtained were investigated by Yang et al. using different media including citrus waste solution (CWS). Figure 61.6 shows SEM images of the bacterial cellulose obtained in the two different media. A seen from the images, fibrils were arranged randomly and the citrus medium promoted fibers that were much thicker than those produced using the HS medium [13Yan1].