Chitin, Chitosan, and Alginate Fibers

Keywords

Chitosan • Ionic solvent • Wet spinning • Dry spinning • Chemical modification • Chitosan • Blend fiber • Chitosan fiber cross-linking

Chitosan has been extensively studied for the production of fibers, and the fibers developed have been thoroughly characterized for their structure, properties, and potential applications. One of the major advantages of using chitosan for fiber production is the solubility of chitosan in common solvents that are relatively inexpensive and environmentally friendly. Table 25.1 lists the most common solvents that have been studied for dissolving chitosan. In addition to the solvents, several other parameters have also been reported to influence the properties of chitosan fibers produced. El-Tahlawy and Hudson studied the effect of various spinning parameters on the production and properties of chitosan fibers [06El]. They reported that viscosity of the solution was critical for fiber production and that adding salt such as sodium acetate assisted in controlling the viscosity, draw ratio, and therefore fiber properties. Similarly, it was reported that the process used to dry the fibers after coagulation also influenced fiber properties. Drying in a methanol coagulation bath provided fibers that could easily separate from each other and have a smooth surface and higher mechanical properties than direct, radiant, or forced air heating [98Kna]. The effect of demineralization time and temperature on the properties and biodegradation of chitosan fibers was investigated by Judawisastra et al. [12Jud]. It was reported that demineralization caused degradation of the polymers and led to an increase in the diameter of the fibers, reduced tenacity by 52 %, and increased elongation (136 %). Biodegradation of the fibers in a phosphate-buffered solution containing 2 % lysozyme increased by 17 %. Similarly, ripening of chitosan dissolved in acetic acid was found to substan­tially affect fiber properties [03Lee]. Increasing ripening time continually decreased tenacity and modulus but increased elongation. Thermal analysis showed that the

peak temperature and thermal degradation temperature decreased with an increase in ripening time.

Regenerated Protein Fibers

Keywords

Honeybee silk • Coiled-coil protein • Transgenic expression • E. coli • Protein component • Fiber properties

Unlike Bombyx mori or spider silks that consist of large repetitive sequences and P-sheets, honeybees secrete four different types of small coiled-coil proteins in nearly equal proportions with a molecular weight of about 30 kDa [08Shi, 10Wei]. These proteins are non-repetitive and are rich in alanine residues, and the proteins were found to be stable in water [08Shi]. Due to their lower molecular weights and unique structure, it was supposed that honeybee silks could easily produce recombinant proteins. To examine this, four proteins (ABS 1-4 with 315, 289, 317, and 321 residues) from the Asiatic honeybee were expressed in Escherichia coli, and the structures of the proteins were studied. The yield of proteins in E. coli was 30, 30, 10, and 60 mg/mL for ABS-1-4, respectively. Corresponding molecular weights obtained for the proteins were 55, 32, 38, and 50 kDa, respectively. Proteins generated were found to have about 65 % coiled-coil sequences but with low (9-27 %) a-helix content and high% (45-56 %) of random coils. In addition, about 26-35 % of p-sheets were also discovered [08Shi]. Some of the properties of the recombinant honeybee silk proteins expressed in E. coli are listed in Table 51.1. Overall, it is seen that the recombinant production of honeybee silks was unable to generate the secondary and tertiary structure seen in native honeybee silk [10Wei]. In the native honeybee silks, the four isolated proteins are found as a complex but have weak interactions between them. In solution, a-sheets, P-sheets, and random coils coexist depending on the pH of the solution. The presence of high amounts of alanine that provides limited hydrophobic interactions was suggested to be the reason for the inability of the silks to maintain higher levels of a- or p-helices [08Shi]. In a similar study, recombinant proteins with yields between 0.5 and 2.5 g/L were obtained using honeybees (Apis mellifera), and the proteins were formed into fibers [10Wei]. Four of the distinct honeybee proteins

were expressed in E. coli (Rosetta 2 DE3), and proteins were collected. Proteins containing all four components were concentrated to get the required viscosity, and the solution was manually drawn into fibers between the prongs of tweezers. Fibers were coagulated in 90 % methanol and 10 % water bath and drawn to about 2x the length and air-dried. Tensile properties of the fibers obtained are listed in Table 51.2.

The as-drawn fibers were soluble in water but became insoluble after coagula­tion in methanol indicating the transition from coiled coil to p structure. Drawing the fibers resulted in an alignment of the proteins and a substantial improvement in tensile properties as seen in Table 51.3. After drawing (1x), fibers had similar elongation and modulus but lower strength than native silk fibers. It was suggested that protein in honeybee glands pre-assembles into aligned liquid crystals before spinning. However, this notion has been proven to be a non-requisite for spider silks as discussed in an earlier chapter in this part. Further studies were done to under­stand the role of the four components in honeybee silks on fiber properties and to investigate if a single protein component was able to replicate the fiber properties obtained using a combination of all the four components [11Sut]. Of the four components, AmelF3 was found to be most suitable for fiber formation since it remained in solution even at high concentrations required for fiber formation, whereas the other components precipitated. Fibers were extruded from the AmelF3 fraction and from the combined AmelF1-4 components and drawn in a methanol bath. Properties of the fibers obtained from AmelF3 and the combined proteins are shown in Table 51.3. As seen in the table, fibers produced from AmelF1-4 had higher strength and lower extensibility when drawn to the same extent. However, AmelF3 will be about 50-60 % stronger than AmelF1-4 if fibers of equivalent diameters are considered. As with other silk fibers, drawing was found to increase the p-sheet content leading to increased tensile strength. It was suggested that the recombinant protein fibers consisted of coiled-coil structures

that were linked by p-sheets. The extent of alignment of p-sheets could be con­trolled during fiber formation. Fibers with higher amounts of coiled-coil structure with low drawing resulted in fibers with moderate strength and toughness, whereas fibers with higher levels of p-sheets were found to have higher strength but low extensibility [11Sut]. In a further continuation of their research, fibers were pro­duced from AmelF3 and then heated at 190 °C for 1 h to covalently cross-link the proteins [13Poo]. Fibers (34 pm) obtained had strength of 1.4 g/den and elongation of 42 % and were stable in protein denaturants such as urea and guanidium. These fibers were knitted and woven into a tubular sheet shown in Fig. 51.1 [13Poo].

References

[08Shi] Shi, J., Lua, S., Du, N., Liu, X., Song, J.: Biomaterials 29, 2820 (2008)

[10Wei] Weisman, S., Haritos, V. S., Church, J. S., Huson, M. G., Mudie, S. T., Rodgers, A. J.W., Dumsday, G. J., Sutherland, T. D.: Biomaterials 31, 2695 (2010)

[11Sut] Sutherland, T. D., Church, J. S., Hu, X., Huson, M. G., Kaplan, D. L., Weisman, S.: PLoS One 6(2), 1 (2011)

[13Poo] Poole, J., Church, J. S., Woodhead, A. L., Huson, M. G., Sriskantha, A., Kyaratzis, I. L., Sutherland, T. D.: Macromol. Biosci. 13, 1321 (2013)

Biocomposites from Renewable Resources

Keywords

Biocomposite • Renewable resource • Agricultural byproduct • Matrix • Rein­forcement • Synthetic biopolymer • Protein • Matrix

The term “biocomposites” has been widely used to denote composites that are made using either the matrix or reinforcement or both from renewable resources that are biodegradable. Conventionally, biocomposites were developed using natural cellu­lose fibers such as jute and flax as reinforcement to replace glass fibers with polypropylene, polyethylene, epoxy, and other synthetic polymer-based matrices. The advent of biopolyesters such as poly(lactic acid) and poly(hydroxy alkoanates) led to a quantum jump in the research on developing biocomposites using both the matrix and reinforcement from renewable resources. In addition, efforts were made to utilize agricultural by-products such as corn stover, wheat straw, and coir fibers as reinforcement resulting in inexpensive and renewable composites. However, biopolyesters such as poly(lactic acid) are considerably more expensive and also do not have the performance properties comparable to that of the traditional synthetic polymers such as polypropylene and polyethylene. Therefore, resins/matrices have also been developed from agricultural byproducts. For instance, soy proteins and wheat gluten have been used as matrix in their native form and also after various chemical modifications.

There is an infinite amount of literature on biocomposites and it is not feasible to provide an exhaustive review of available literature in one part. Several authorita­tive books on biocomposites have been published. The focus of this part is to provide an overview of the possible sources and properties of biocomposites developed using renewable resources that have been covered in the previous parts. Particular emphasis has been on completely biodegradable composites but some examples of composites containing either the matrix or reinforcement obtained for renewable resources have also been included for illustrative purposes. In some cases, the reinforcing fibers have been derived from renewable and biodegradable agricultural residues but have been combined with traditional syn­thetic polymers. Similarly, composites have been developed by directly blending agricultural residues such as corn stover or wheat straw into synthetic polymer or protein-based matrices that have been derived from renewable resources. Such literature has also been reviewed. Overall, the purpose of this part is to provide an overview of the composites developed from agricultural by-products and coproducts and the properties and potential applications of such composites.

Although efforts have been made to divide the part into clear and distinct chapters, some overlap is inevitable. For instance, lignocellulosic fibers have been used as reinforcement with both synthetic polymers and biodegradable polymers as matrix. Therefore, some of the reinforcement and matrix materials have been covered in multiple chapters but the literature reported has not been duplicated.

Natural Cellulose Fibers from Renewable Resources

Keywords

Banana fiber • Pseudo-stem • Fiber extraction, fiber yield • Mechanical treatment

One of the most ubiquitous fruits, banana is widely grown across the world. About 120-150 million tons of bananas are grown annually in the world, and it is the fourth most important food product in the world. However, the banana fruit only represents about 12 % of the weight of the plant and the stem; leaves and other parts are not generally edible. Therefore, efforts have been made to use banana leaves and stems for various nonfood applications including fiber production. Fibers are obtained from the pseudo-stem of the plant mostly by mechanical means. Full — fledged banana fiber production has been reported to be operational in several countries. Some of the products developed from banana fibers include textiles, paper, floor mats, and composites. In terms of properties, banana fibers have the typical composition of fibers obtained from lignocellulosic by-products and contain about 50 % cellulose, 17 % lignin, and 4 % ash [09Gui]. However, the composition of the banana fibers reported varies widely, and fibers with lignin content as high as 17 % have been reported [08Hab]. In addition to the stem, fibers have also been obtained from the leaf and rachis of the banana plant. Considerable variations in the tensile properties were observed for the fiber bundles obtained from the different parts and also depending on the method of extraction as seen in Table 7.1 [08Gan]. Tensile properties of the fibers obtained from the banana stems are similar to those of common lignocellulosic fibers such as jute, but the elongation is considerably lower than that of the coconut and palm (Borassus flabellifer) fibers. Low elongation of the banana fibers should mainly be due to the lower microfibril­lar angle (11°) and relatively high % crystallinity [08Muk]. Banana fibers also appear to have a hollow center similar to that found in a few other natural cellulose fibers. Considerable variation in the tensile properties, especially elongation, was observed for fibers with various diameters (50-250 pm) as seen in Tables 7.1, 7.2, and 7.3 [10Ven]. In addition to the stems, fibers have also been obtained from the

leaves of the banana plant. Typically, banana plants produce about 30 leaves as long as 2 m and 30-60 cm wide [07Bil]. Fibers obtained from banana leaves had about 26 % cellulose, 17 % hemicellulose, and 25 % lignin, but the fiber properties are not reported [07Bil]. A Switzerland-based company (Swicofil) advertises that it had developed fabrics from ring — and rotor-spun banana fibers. Ring-spun yarns in counts ranging from Ne 8/1 to 40/1 and rotor-spun yarns with counts (Ne) ranging from 8/1 to 30/1 were reported to be available in 100 % form and also as blends with cotton, modal, Tencel, and soy protein fibers. Banana fibers are reported to be available on the market for about US$0.43-0.81/kg compared to $0.15-0.60 for hemp and $0.15-$0.21/kg for flax.

References

[83Kul] Kulkarni, A. G., Satyanarayana, K. G., Rohatgi, P. K., Vijayan, K.: J. Mater. Sci. 18, 2290 (1983)

[07Bil] Bilba, K., Arsene, M., Ouensanga, A.: Bioresour. Technol. 98, 58 (2007)

[07Rao] Rao, M. M.K., Rao, M. K.: J. Comp. Struct. 77, 288 (2007)

[08Gan] Ganan, P., Zuluga, R., Restrepo, A., Labidi, J., Mondragon, I.: Bioresour. Technol. 99, 486 (2008)

[08Hab] Habibi, Y., El-Zawawy, W. K., Ibrahim, M. M., Dufresne, A.: Compos. Sci. Technol. 68, 1877 (2008)

[08Muk] Mukhopadhyay, S., Fangueiro, R., Arpac, Y., Senturk, U.: J. Eng. Fibers Fabr. 3(2), 39 (2008)

[09Gui] Guimaraes, J. L., Frollini, E., da Silva, C. G., Wypych, F., Satyanarayana, K. G.: Ind. Crop Prod. 30, 407 (2009)

[10Das] Das, P. K., Nag, D., Debnath, S., Nayak, L. K.: Indian. J. Tradit. Knowl. 9(2), 386 (2010)

[10Ven] Venkateshwaran, N., Elayaperumal, A.: J. Reinf. Plast. Compos. 29(15), 2387 (2010)

[13Bua] Buana, S. A.S., Pasbaskhsh, P., Goh, K. L., Bateni, F., Haris, M. R.H. M.: Polymers 14

(4), 623 (2013)

Chitin, Chitosan, and Alginate Fibers

Keywords

Alginate • Polysaccharide • Blend • Konjac glucomannan • Absorption

Calcium alginate fibers intended for wound dressing applications were mixed with another polysaccharide branan ferulate which is a recognized polymer for wound dressing and treating ulcers and sores [03Mir]. The influence of the alginates supplied by different companies and the addition of ferulate on the mechanical properties were studied. Up to 75 % ferulate could be added to selected types of alginates without sacrificing the tensile properties. Dry tenacities of the fibers varied from 0.2 to 1.6 g/den, and elongation was between 10 and 40 %. In a similar research, calcium alginate fibers were blended with konjac glucomannan (KGM) and later treated with silver nitrate to impart antimicrobial activity, and the properties of the blend fibers were studied. The addition of KGM increased the dry strength but decreased the wet strength. It was suggested that KGM and alginate had good compatibility, and the addition of silver imparted good antimicrobial activity [07Fan]. In this research, dry tenacity of the pure alginate fiber was 1.2 g/den, and elongation was 18 %. The addition of KGM increased the strength up to 1.6 g/den and elongation up to 34 %. Substantial increase in water retention was seen with the retention value being 1,000 % with 70 % KGM compared to 91 % without KGM. Fibers treated with silver had higher than 99.99 % bacterial reduc­tion to S. aureus. Wet strength of the fibers varied between 0.04 and 0.3 g/den, considerably lower than the dry strength.

Calcium alginate fibers were dyed with acid dyes (acid red 249, acid yellow 117, and acid blue 80), and the changes in color fastness and tensile properties were evaluated [12Lv]. All three dyes showed high dye exhaustion between 70 and 85 % and also had good fastness to washing and rubbing. Dyeing did not result in degradation of the fibers with only about 11-13 % weight loss. Calcium alginate fibers were converted into alginic acid and later into sodium alginate by treating the fibers in hydrochloric acid and sodium hydroxide, respectively [06Qin], in order to improve their gelling ability and absorption capacity. It was reported that converting calcium alginate into sodium alginate fibers and fabrics made them more absorbent than calcium alginate and alginic acid fibers.

References

[03Mir] Mirafatab, M., Qiao, Q., Kennedy, J. F., Anand, S. C., Groocock, M. R.: Carbohydr. Polym. 53, 225 (2003)

[06Qin] Qin, Y., Hu, H., Luo, A.: J. Appl. Polym. Sci. 101, 4216 (2006)

[07Fan] Fan, L., Zhu, H., Zheng, H., Xu, Y., Zhang, C.: J. Appl. Polym. Sci. 106, 3903 (2007) [12Lv] Lv, F., Zhu, P., Wang, C., Zheng, L.: J. Appl. Polym. Sci. 126, E382 (2012)

Single nanofibers of chitosan, collagen, and their blends were produced and the interactions between collagen and chitosan and the properties of the fibers obtained were evaluated [08Che1]. Collagen and chitosan were dissolved using HFIP or trifluoroacetic acid and electrospun at room temperature. A new electrospinning arrangement was done to collect single nanofibers for tensile testing. Single fibers with diameters of several micrometers were obtained but it was not possible to obtain single nanofibers. However, the electrospun matrix produced had fibers in the nanometer range. Tensile properties of the single electrospun fibers with various ratios of chitosan are given in Table 58.5 [08Che1]. As seen in the table, increasing the amount of chitosan increased the strength and modulus up to 50 %. Interest­ingly, the elongation of the fibers is considerably low except when the chitosan ratio was 20 % at which the elongation was as high as 46 %. The changes in the tensile properties of the membranes were attributed to the interchanges in the intermolec­ular interactions between chitosan and collagen.

To combine antimicrobial and cell adhesion properties, chitosan was blended with gelatin and electrospun into nanofibrous scaffolds for skin tissue engineering [10Dha]. Fibers with diameters between 120 and 220 nm were obtained using 50/50 ratio of chitosan and gelatin. Average tensile strength of the 50/50 blend scaffolds was 26 MPa, significantly higher than that of the matrices developed from the individual polymer. Blend scaffolds were suggested to have strength similar to that of skin.

Single fibers and membranes were formed from blends of collagen-chitosan by electrospinning and their mechanical properties were investigated [09Che]. Mechanical properties were found to be dependent on the diameter of the fibers and the ratio of collagen to chitosan in the fibers. Fibers with smaller

Table 58.5 Properties of electrospun single chitosan-collagen fibers with different chitosan contents [08Chel] Chitosan (%) 0 20 40 50 60 80 Diameter (pm) 7.8 ±5.0 7.4 ±1.2 6.8 ± 12.1 5.6 ±4.0 3.4 ±0.8 9.1 ±1.4 Strength (MPa) 23.7 ±5.8 21.7 ±19.3 62.8 ±14.1 61.8 ±26 47.0 ±19.1 10.5 ±8.0 Elongation (%) 2.6 ±1.4 46.0 ± 23 1.2 ±0.4 1.3 ±0.8 1.3 ±0.7 0.4 ±0.04 Modulus (MPa) 1,371 ±225 1,611 ±793 5,966 ±2,137 6,801 ±3,256 4,159 ±1,195 3,601 ±485

diameters had higher strength but low elongation. Single fibers from the chitosan- collagen blend had average diameters ranging from 434 to 691 nm and had strength between 20 and 60 MPa, elongation between 5 and 50 %, and modulus between 1 and 7 GPa depending on the chitosan content in the blend.

Regenerated Cellulose Fibers

Keywords

Cellulose dissolution • Low temperature • Urea-sodium hydroxide • Fiber cross section • Sol-gel process

An extension of the alkali system of dissolving cellulose and the most recent development in the production of regenerated cellulose fibers has been the dissolu­tion of cellulose using NaOH/urea or NaOH/thiourea systems [04Rua, 06Che]. In one such approach, cotton linter pulp (DP ~ 550) of 4-5 wt% was dissolved using NaOH (9.5 %) and thiourea (4.5 %) solution that was precooled to —8 to —10 °C [01Zha, 10Zha]. After dissolution, the solution was filtered, degassed, and extruded through a spinneret into a coagulation bath. Various chemicals (mainly acids or salts) were added into the coagulation bath, and it was found that aqueous solutions of sulfuric acid, hydrochloric acids, acetic acid, or ammonium salts were best for fiber formation. Fibers were produced using a laboratory wet spinning system at a pressure of 0.15 MPa and with a spinneret diameter of 0.12 mm [04Cai, 06Che]. Morphologically, the fibers obtained had a circular cross section contrary to traditionally produced viscose fibers that have a distinguishing irregular cross section. Unlike the conventional viscose process where complete dissolution of cellulose occurs, the new solvent system is considered to be a physical sol-gel process that helps to retain the circular shape of the fibers [04Rua]. Some of the properties of the fibers obtained using the NaOH/urea systems are compared to the traditional viscose fibers obtained from the NMMO system in Table 17.1.

The degree of polymerization (DP) of the fibers obtained from the NaOH/urea system was similar to that of the DP of the cotton linters used for fiber production indicating that there was no significant degradation of cellulose during the dissolu­tion using the new system. Tensile properties of the fibers were similar to that of the traditional viscose and cuprammonium fibers but lower than that of the fibers obtained using the NMMO system mainly because of the better drawing of the fibers during the commercial-scale NMMO production process. Recently, the

Table 17.1 Comparison of the properties of regenerated cellulose fibers obtained through the NaOH/urea system compared to the traditional viscose process and the NMMO process

industrial-scale production of regenerated cellulose fibers using the NaOH/urea system has been reported [10Li]. Fibers with strength (2.0 ± 0.2 g/den) and elonga­tion of 19 %, similar to that of commercial rayon, were obtained. Figure 17.1 shows the digital images of the dissolution and fiber production process, and Fig. 17.2 shows the images of the actual fibers and cross section of the fibers obtained [10Li]. Although fibers with properties similar to those commercially available were produced using the NaOH/urea system, there is an upper limit on the degree of polymerization and molecular weights of cellulose that can be dissolved using this

system [08Wan]. For instance, a 6 % NaOH/4 % urea aqueous solution could only completely dissolve cotton linters with Mw of up to 6.7 x 104 g/mol and cellulose II with Mw up to 11.2 x 104 g/mol [08Wan]. To overcome this limitation, enzymatic pretreatment was used to promote the dissolution of cellulose with high molecular weight. Increase in cellulose solubility from 30 to 65 % and shortening of dissolu­tion time were observed after the enzymatic treatment [08Wan]. Unlike the NaOH/ urea treatment which breaks inter — and intra-cellulose bonds, enzyme treatment attacks and cuts the cellulose crystal and increases the accessibility to NaOH and urea solutions and therefore allows the use of high molecular weight cellulose [08Wan].

Properties of the fibers obtained from the NaOH/urea system were heavily dependent on the conditions used for cellulose dissolution. As seen in Fig. 17.3, increasing the concentration of NaOH continually increased the solubility of cellulose up to an NaOH concentration of 8 %. Similarly, increasing temperature above —10 °C considerably decreases dissolution as seen in Fig. 17.4. A phase transition between gel formation and solution form occurs with the change of the temperature and concentration as seen in Fig. 17.5.

Instead of using pure cellulose, attempts were also made to use modified cellulose for dissolution and production of fibers using the NaOH/urea method of

dissolution. In one such attempt, hydroxyethylated cellulose (HEC) with low levels of substitution was dissolved in alkali solutions and extruded into fibers [13Wan]. HEC was added into 8 % NaOH/8 % urea and 6.5 % thiourea solution and cooled to —10 °C. The solution was stirred at 0 °C for 2 h to dissolve the cellulose. Dissolved cellulose was extruded into a coagulation bath consisting of 12 % sulfuric acid and 10 % sodium sulfate to precipitate the fibers which were later washed and dried [13Wan]. Structure and properties of the regenerated cellulose fibers obtained from HEC were considerably different compared to those obtained using unmodified cellulose through the traditional processes as seen in Table 17.2. The original cellulose seen in Fig. 17.6a had the cellulose I crystal structure, whereas the etherified cellulose (HEC) in Fig. 17.6b and the fibers obtained from HEC (Fig. 17.6c) had the typical cellulose II structure. The comparison of the properties between the HEC fibers and regenerated fibers obtained using the lyocell

process shows that etherification resulted in considerable decrease in % crystallinity and the fibers were also much weaker compared to the regenerated cellulose fibers obtained using the lyocell process [13Wan].

References

[01Zha] Zhang, L., Ruan, D., Zhou, J.: Ind. Eng. Chem. Res. 40, 5923 (2001)

[04Cai] Cai, J., Zhang, L., Zhou, J., Li, H., Chen, H., Jin, H.: Macromol. Rapid Commun. 25,

1558 (2004)

[04Rua] Ruan, D., Zhang, L., Zhou, J., Jin, H., Chen, H.: Macromol. Biosci. 4, 1105 (2004)

[06Che] Chen, X., Burger, C., Fang, D., Ruan, D., Zhang, L., Hsiao, B. S., Chu, B.: Polymer 47,

2839 (2006)

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

[10Li] Li, R., Chang, C., Zhou, J., Zhang, L., Gu, W., Li, C., Liu, S., Kuga, S.: Ind. Eng. Chem. Res. 49, 11380 (2010)

[10Zha] Zhang, S., Li, F., Yu, J., Hsieh, Y.: Carbohydr. Polym. 81, 668 (2010)

[13Wan] Wang, W., Zhang, P., Zhang, S., Li, F., Yu, J., Lin, J.: Carbohydr. Polym. 98, 1031 (2013)

Natural Protein Fibers

Keywords

Mussel • Anchoring thread • Rigid fiber • Flexible fiber • Collagen distribution

Marine animals such as mussels produce fibrous attachments generally called byssus as shown in Fig. 41.1. Each thread in a byssus is about 2-3 cm long and about 100-200 qm in diameter [13Lin]. Byssal threads were reportedly woven into fabric in Greece to produce fine clothing [07Ald]. These byssal threads have extraordinary structural arrangement and properties not seen in other protein fibers. The thread consists of two regions, the distal portion (threads) which is rigid and stiff and the proximal region (threads) that is approximately 50-fold less stiff than the distal threads due to the unique composition and structure of the proteins in the threads [01Vac].

Tensile properties of the mussel threads have been examined under various conditions. Table 41.1 provides a comparison of the tensile properties of the distal mussel threads treated in different conditions. As seen in the table, dehydrated threads had higher stiffness, whereas the hydrated threads were more flexible and had high elongation. It was suggested that in addition to the preCol domains in the fibers, the adjacent domains assist in load dissipation and are crucial for the load­bearing ability of the threads [11Hag]. Recent studies have suggested that the high strength of the byssal threads is mainly due to the combination of the stiff and soft regions (distal/proximal) in 80/20 ratio [13Qin]. Other researchers have also reported tensile stress of 0.5 g/den for fresh hydrated distal mussel threads and 0.6 g/den for aged and hydrated threads [07Ald]. Corresponding values for the dehydrated threads were 0.7 g/den, respectively. The ability of the threads to recover from stretching in water with various chemicals was studied by Vaccaro and Waite [01Vac]. Table 41.2 shows the Young’s modulus and energy dissipated by distal byssal threads after three stress-strain cycles. Chemical treatments decreased the modulus and energy dissipation of all the fibers. EDTA-treated fibers

Table 41.1 Comparison of the properties of the mussel distal threads treated in artificial seawater and distilled water

showed different behavior than the other proteins due to the chelating action and stabilization of the proteins.

In terms of composition, proximal threads consist of oriented collagen — containing fibrils dispersed in a soft proteinaceous matrix, whereas the distal threads are composed of packed fibrous bundles (Fig. 41.2) held together by covalent and non-covalent bonds. The matrix consists of glycine-, tyrosine-, and

Fig. 41.2 Schematics of the distribution and properties of byssal threads. From Harrington and Waite [09Har]

asparagine-rich proteins that have a unique repeated sequence motif and are distributed throughout the thread [09Sag]. Such an arrangement provides the rigidity and strength required for the threads to adhere to surfaces and resist the external forces and at the same time provides flexibility required to move and capture prey [01Vac, 11Hag]. Threads are about 95 % protein and consist of fibrils composed of collagen embedded in a protein matrix surrounded by a granular cuticle as shown in Fig. 41.3 [11Hag]. Further investigations have shown that the threads are composed of collagen and are termed PreCol (preCol-D, — NG, and — P) that forms 96 % of the distal region and 66 % of the proximal part [08Har, 09Har, 13Arn]. Protein chains (motifs) in preCol-D resemble silk, in preCol-NG resemble glycine-rich plant cell wall proteins, and that of preCol-P are similar to that of elastin. The composition and arrangement of these three PreCol components vary across the length of the fibers, and this distribution is crucial in determining the properties of the thread. Some studies have suggested that the preCol-NG serves as a mediator between preCol-D and preCol-P and responsible for the gradient properties seen in the fibers [98Qin]. A schematic of the distribution of the PreCol components is shown in Fig. 41.3b.

Hagenau et al. have shown that the distal region is composed of high levels of P-sheets that are well oriented, whereas the proximal part is less oriented and mainly consists of a-helices [09Hag]. X-ray diffraction studies (Fig. 41.4) have clearly shown that the distal portion is well oriented, the proximal portion is less oriented, and the middle portion has an orientation in between that of the distal and proximal regions. Further analysis of these portions using FTIR revealed that the proximal portion is composed of proteins with about 47 % a-helices, 15 % p-sheets, 25 % of aggregate sheets, and 13 % of triple helix structures. Comparatively, the distal portion was composed of 70 % p-sheets, 22 % triple helices, and 8 % of aggregate sheets [09Hag]. However, it was suggested that uncertainties existed in the prediction of the structure of the Byssal threads using X-ray diffraction and FTIR studies due to the presence of mixture of proteins. SEM image of a distal byssal thread in Fig. 41.5 shows a dense fibrillar network inside a proteinaceous matrix.

References

[98Qin] Qin, X., Waite, J. H.: Proc. Natl. Acad. Sci. USA 95, 10517 (1998)

[01Vac] Vaccaro, E., Waite, J. H.: Biomacromolecules 2, 906 (2001)

[07Ald] Aldred, N., Wills, T., Williams, D. N., Clare, A. S.: J. R. Soc. Interface 4, 1159 (2007) [08Har] Harrington, M. J., Waite, J. H.: Biomacromolecules 9, 1480 (2008)

[09Hag] Hagenau, A., Scheidt, H. A., Serpell, L., Huster, D., Scheibel, T.: Macromol. Biosci. 9, 162 (2009)

[09Har] Harrington, M. J., Waite, J. H.: Adv. Mater. 21, 440 (2009)

[09Sag] Sagert, J., Waite, J. H.: J. Exp. Biol. 212, 2224 (2009)

[11Hag] Hagenau, A., Papadopoulos, P., Kremer, F., Scheibel, T.: J. Struct. Biol. 175, 339 (2011)

[13Arn] Arnold, A. A., Byette, F., Seguin-Heine, M., LeBlanc, A., Sleno, L., Tremblay, R., Pellerin, C., Marcotte, I.: Biomacromolecules 14, 132 (2013)

[13Lin] Lintz, E. S., Scheibel, T. R.: Adv. Funct. Mater. 23, 4467 (2013)

[13Qin] Qin, Z., Buehler, M. J.: Nat. Commun. 4, 2187 (1998)

Bacterial cellulose is primarily produced using various sugars as feedstock conven­tionally using static culture and also by using agitated culture. Typical production rates of bacterial cellulose that have been reported are 5-8 g/l using fructose and corn syrup and 7-9.2 g/l using stirred tank bioreactors. Instead of using single sugars as feedstocks, Dahman et al. investigated the ability to produce bacterial cellulose from multiple sugars. Sugar compositions similar to those found in acid hydrolysates of some agricultural residues were utilized with Gluconacetobacter xylinus as the bacteria [10Dah]. Under identical conditions, the bacterial production from single sugars varied from 1.1 to 5.7 g/l compared to 2.4 to 5.2 g/l for the mixed sugars. Table 61.2 lists the type of sugar feedstock used and the yield of bacterial cellulose obtained. It was concluded that agricultural residues could be potential feedstocks for biocellulose nanofiber production.

In a similar approach, elephant grass (Pennisetum purpureum) was used as a feedstock [13Yan2]. Acid hydrolyzed and detoxified elephant grass was inoculated with bacteria for 14 days under static fermentation. About 60 % of sugar was converted into bacterial cellulose and the yield was about 6.4 g/l. Morphologically, the bacterial cellulose transformed from an initial dense pellicle into microfibrils after 8 days of fermentation. Figure 61.7 shows scanning electron microscopy (SEM) images of the formation of the bacterial cellulose fibers after different stages of fermentation with fibrils obtained having diameters between 15 and 100 nm. X-ray diffraction patterns of the cellulose showed diffraction peaks typical of

cellulose I with % crystallinity of the fibers increasing from 23 to 54 % after 2 weeks of fermentation. After the final fermentation, cellulose crystals that had a size of about 87 A were found to have a crystallinity index of up to 99 %.

Similar to using elephant grass for bacterial cellulose production, wheat straw was pretreated with [AMIM]Cl and later enzymatically hydrolyzed to obtain sugars. After fermentation, the bacterial cellulose obtained had cellulose I crystal form with a yield of 8.3 g/l [13Che1]. Crystallinity of the straw used was 49 % which decreased to 35.9 % after pretreatment with ionic liquids which promoted growth of bacterial cellulose. In another report, bacterial cellulose was developed from canola straw for potential reinforcement for paper [13You]. Bacterial cellulose nanofibers obtained had an average diameter of 45 nm, crystallinity of 80 % with crystal size being 6.2 nm. Tensile strength of the BC nanofibers was 1.4 g/den and modulus was 133 g/den when measured using a tensile tester. Figure 61.8 shows images of the bacterial cellulose produced, and the average

diameter of the fibers can be deduced from the SEM image [13You]. When the bacterial cellulose was made into paper, the specific strength was 142 Nm/g, substantially higher than the paper made from ground cellulose or micropaper obtained from the same wheat straw. Similarly, a higher burst strength was also observed for the bacterial cellulose paper. To decrease the cost of producing bacterial cellulose, wheat straw was hydrolyzed using dilute acid hydrolysis and the hydrolysate obtained was used as feedstock. However, detoxification of the straw hydrolysate was necessary to obtain good yields [11Hon]. A considerably high yield of 15.4 mg/l was obtained from the wheat straw hydrolysate.

To overcome the lack of antimicrobial activity, bacterial cellulose nanofiber membranes were surface functionalized with aminoalkyl groups using 3-aminopropyltrimethoxysilane. Treated membranes were found to have excellent antimicrobial activity to Staphylococcus aureus and Escherichia coli and were also nontoxic to adipose derived mesenchymal stem cells and therefore considered to be useful for biomedical applications [13Fer]. A marginal increase in the strength and elongation of the cellulose nanofiber mats was observed with strength being about 6 MPa, elongation of 1.2 %, and modulus of 3.6 GPa [13Fer].

Bacterial cellulose filaments obtained from Gluconacetobacter xylinum and Hestrin-Schramm medium were tested to determine their Young’s modulus using Raman spectroscopy, and mechanical properties of the bacterial cellulose sheet were determined using an Instron tester [08Hsi]. Cellulose sheets had a modulus of

9.1 GPa, tensile strength of 240 MPa and elongation of 2.6 %. To determine the modulus of a single filament, the shift in the position of the peak at 1,095 cm-1 corresponding to the C-C or C-O bond stretching was observed. Changes in the position of the peaks were correlated to strain and the modulus of the single filament was determined to be 877 g/den. Other theoretical estimates have reported the modulus of the cellulose sheets to be between 1,046 and 1,192 g/den. Bacterial cellulose nanocrystals obtained from Gluconacetobacter xylinum that are typically

longer (100 nm to several micrometers) were added into silk fibroin solution and electrospun into fibers. The amount of cellulose crystals was varied from 0 to 7 % with corresponding increase in the diameter of the nanofibers from 230 to 430 nm. Table 61.3 shows the mechanical properties of the nanofibers containing various levels of bacterial cellulose nanocrystals. The stability and degradability of the nanofibers were not studied [12Par].

Cotton-based waste textiles were used as feedstock to produce bacterial cellu­lose using Gluconacetobacter xylinum. Before culture, the textiles were dissolved in 1-allyl-3-methylimidazolium chloride and the hydrolysate was used for culture. Yield of bacterial cellulose was 10.8 g/l, much higher than that obtained using glucose-based medium [12Hon]. The bacterial cellulose obtained had a water holding capacity of 99 % and tensile strength was 0.07 MPa [12Hon]. In a similar study prior to the report by Hong et al., colored 100 % cotton and 40/60 polyester/ cotton waste t-shirts were dissolved (concentrated phosphoric acid was found to dissolve 100 % cotton at 50 °C) using various solvents and the fermentable sugars obtained were used to culture Gluconacetobacter xylinus [10Kuo]. Fermentation yields of up to 1.8 g/l were obtained after 7 days of static culture. Figure 61.9 shows image of the cellulose pellicle obtained after 7 days of culture in sugar solution,

hydrolysate obtained from the fabrics, and the discolored hydrolysate [10Kuo]. Waste fiber sludge, a residue obtained during the processing of cellulose by pulp mills and lignocellulosic biorefineries was also used as a source to generate bacterial cellulose [13Cav]. Sludges obtained from the sulfate (SAFS) and sulfite (SIFS) processes were enzymatically hydrolyzed and the resulting hydrolysates were used for BC production. Table 61.4 shows some of the properties of the bacterial cellulose obtained using the two types of sludge and the reference medium. As seen from Table 61.4, the fiber sludge produced bacterial cellulose with properties comparable or superior to that of the reference medium.

Agricultural wastes such as pineapple peel juice and sugarcane juice were also used as culture media to produce bacterial cellulose with a yield of about 2.8 g/l, higher than that produced from the regular medium. Cellulose fibrils obtained had width of 20-70 nm but the formation of the fibrils was hindered due to the presence of other carbohydrates in the juice [11Cas]. Other agricultural residues such as wine and pulp wastes when used as sources for production of bacterial cellulose pro­duced considerably low yields of cellulose in the range of 0.6-0.3 g/l after 96 h of incubation but the yields increased by about 100-200 % for crude glycerol and grape skins when organic and inorganic nitrogens were added [11Car].

In a similar approach, waste beer yeast (WBY) was used for production of bacterial cellulose from the strain Gluconacetobacter hansenii CGMXX 3917 after a two-step pretreatment. The WBY was hydrolyzed using four different approaches and the hydrolysate obtained was directly used to produce bacteria [14Lin]. A highest bacterial cellulose yield of 7 g/l, six times higher than that obtained from untreated WBY, was achieved using WBY treated by ultrasonication [14Lin]. A new bacterium (Gluconacetobacter sp. F6) was isolated from rotten fruit and the conditions required to obtain optimum cellulose from the fruit waste were studied [12Jah]. In addition to rotten fruit, soil, vegetables, and vinegar were also studied as potential sources for bacterium. A cellulose yield of

4.5 g/l was obtained under the optimum conditions of pH 6, temperature of 30 °C, and using glucose as the carbon source [12Jah]. A thick leathery pellicle formed during production of grape wine was studied for its structure and properties and identified as bacterial cellulose from the Gluconacetobacter sp. strain. Films of 25 qm thickness were found to have an unusually high tensile strength of 41 MPa and elongation of 0.98 mm. The films had low oxygen permeability but high water

permeability [11Ran]. Figure 61.10 shows an SEM image of the bacterial cellulose generated by the cells.

Atomic force microscopy (AFM) of bacterial cellulose revealed that Gluconace- tobacter xylinus was reported to synthesize bacteria in the form of fine ribbons, similar to a three dimensional knitted structure. To improve the surface properties of the bacterial cellulose fabric, bacteria was cultured on nine different types of fabrics. Bacterial cellulose showed higher affinity for cotton and viscose compared to wool, silk, or the common synthetic polymers. Among the different fabrics studied, viscose rayon was found to be coated on both sides and such composite fabrics were expected to be suitable for medical applications [12Miz].

Instead of the traditional wet spinning approach of producing fibers from chitosan, a pseudo-dry spinning approach was used by Notin et al. Deacetylated chitosan (degree of acetylation, 2.7 %) with molecular weight of 540,000 g/mol was dissolved using aqueous acetic acid solution. After extrusion, the fibers were exposed to gaseous ammonia rather than coagulating the fibers for precipitation. Upon exposure to ammonia, ammonium acetate was produced that could be easily eliminated. This approach of pseudo-dry spinning did not result in severe damage to the molecular weight of chitosan even after storing the fibers for 2 months. SEM images showed that the chitosan was completely coagulated, and the fibers obtained had a smooth and regular surface. Fibers produced had tenacity between 0.9 and 5 g/den, elongation between 4 and 9 %, and modulus between 238 and 531 g/den [06Not1, 06Not2]. Further studies by the authors showed that post-drying of the fibers for 1 week under ambient atmosphere was necessary to stabilize the fibers [06Not1, 06Not2]. Optimizing the jet-stretch ratio during coagulation and the post­drying of the fibers lead to the production of fibers with a tenacity of 2 g/den and a modulus of 82 g/den. Aging of fibers under ambient condition was reported to assist the formation of anhydrous crystalline form with fibers containing large amounts of tendon forms and lesser amounts of annealed/anhydrous form. X-ray studies showed that the crystallinity index increased with aging time from 15 to 24 % after 5 months of aging. Morphologically, the fibers obtained had a fibrillated surface with an average fibril diameter of 140 ± 50 nm and between 100 and 175 nanofibrils/4 pm2 as seen in Fig. 25.1a, b. Although the fibers produced with this method had better tensile properties compared to fibers produced from other methods, it should be noted that the wet stability of the fibers was not reported. Chitosan fibers are inherently weak when wet and need to be cross-linked or chemically modified to improve stability in aqueous environments [06Not2].