This thesis was motivated by the practical need to develop a scalable and cost-effective separation method to recover hyaluronic acid (HA), a commercially valuable medical biopolymer, from bacterial culture broth. This challenge can potentially be addressed by taking advantage of the polyanionic character of HA. Through the electrostatic interaction with cationic matrix, HA is expected to be recovered. In this thesis quaternary ammonium modified cellulose fibers were used to recover HA directly from the Bacillus subtilis culture.
At the first stage of the studies, two variant of cellulose fibers were prepared by grafting different type of the antibacterial quaternary ammonium compound (QAC). Silane (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) and choline based ionic liquid analogue (sodium hydroxide and 2–chloroethyltrimethylammonium chloride based deep eutectic solvent) were used to quaternize the cellulose fibers surface. Results from elementary analysis, grafting ratio determination by weight measurement and Fourier transform infrared spectroscopy demonstrated that either silane or choline has been successfully grafted onto the surface of cellulose fibers after the chemical modification.
Prior to the adsorption studies, the antibacterial assessments were performed. It was shown that both silane modified cellulose fibers (SMC) and choline modified cellulose fibers (CMC) have antibacterial activity against E. coli and B. subtilis, with the former had better activity due to the presence of long alkyl chain on the quaternary ammonium groups of SMC. The antibacterial activity for E. coli is higher than that for B. subtilis due to the difference in cell wall structures. The feasibility of the antibacterial cellulose fibers on HA adsorption was then explored. Adsorption of HA onto the modified cellulose fibers was carried out at different pHs (3–8) and temperatures (277, 291, 310 K). Adsorption isotherms followed the Langmuir isotherm model and the adsorption was thermodynamically favorable. The maximum adsorption capacity for SMC and CMC was found to be 183.67 mg/g (at pH 4, 310 K) and 351.32 mg/g (at pH 7, 277 K), respectively. The adsorbed HA on SMC could be effectively desorbed as much as 90.11% by 1 N NaCl at pH 7 while changing the pH to 3 was effective to desorb 36.50% of HA from CMC. The adsorption capacity of SMC decreased to 49% after 3 cycles, whereas the capacity of CMC decreased to 89%.
Finally, the capability of the modified cellulose fibers on the recovery of HA directly from B. subtilis culture was demonstrated. SMC was used in this process rather than CMC due to its high desorption efficiency. The antibacterial effect of SMC caused by its strong hydrophobic character also good for HA recovery. One gram of SMC was able to adsorb 14.94 mg of HA directly from the B. subtilis culture. The adsorption capacity of SMC used in this recovery process was much less than in the equilibrium adsorption studies due to the competition adsorption occurred in the culture broth.

This thesis was motivated by the practical need to develop a scalable and cost-effective separation method to recover hyaluronic acid (HA), a commercially valuable medical biopolymer, from bacterial culture broth. This challenge can potentially be addressed by taking advantage of the polyanionic character of HA. Through the electrostatic interaction with cationic matrix, HA is expected to be recovered. In this thesis quaternary ammonium modified cellulose fibers were used to recover HA directly from the Bacillus subtilis culture.
At the first stage of the studies, two variant of cellulose fibers were prepared by grafting different type of the antibacterial quaternary ammonium compound (QAC). Silane (3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride) and choline based ionic liquid analogue (sodium hydroxide and 2–chloroethyltrimethylammonium chloride based deep eutectic solvent) were used to quaternize the cellulose fibers surface. Results from elementary analysis, grafting ratio determination by weight measurement and Fourier transform infrared spectroscopy demonstrated that either silane or choline has been successfully grafted onto the surface of cellulose fibers after the chemical modification.
Prior to the adsorption studies, the antibacterial assessments were performed. It was shown that both silane modified cellulose fibers (SMC) and choline modified cellulose fibers (CMC) have antibacterial activity against E. coli and B. subtilis, with the former had better activity due to the presence of long alkyl chain on the quaternary ammonium groups of SMC. The antibacterial activity for E. coli is higher than that for B. subtilis due to the difference in cell wall structures. The feasibility of the antibacterial cellulose fibers on HA adsorption was then explored. Adsorption of HA onto the modified cellulose fibers was carried out at different pHs (3–8) and temperatures (277, 291, 310 K). Adsorption isotherms followed the Langmuir isotherm model and the adsorption was thermodynamically favorable. The maximum adsorption capacity for SMC and CMC was found to be 183.67 mg/g (at pH 4, 310 K) and 351.32 mg/g (at pH 7, 277 K), respectively. The adsorbed HA on SMC could be effectively desorbed as much as 90.11% by 1 N NaCl at pH 7 while changing the pH to 3 was effective to desorb 36.50% of HA from CMC. The adsorption capacity of SMC decreased to 49% after 3 cycles, whereas the capacity of CMC decreased to 89%.
Finally, the capability of the modified cellulose fibers on the recovery of HA directly from B. subtilis culture was demonstrated. SMC was used in this process rather than CMC due to its high desorption efficiency. The antibacterial effect of SMC caused by its strong hydrophobic character also good for HA recovery. One gram of SMC was able to adsorb 14.94 mg of HA directly from the B. subtilis culture. The adsorption capacity of SMC used in this recovery process was much less than in the equilibrium adsorption studies due to the competition adsorption occurred in the culture broth.

Abbot, A.P., Bell, T.J., Handa, S., Stoddart, B. 2006. Cationic functionalisation of cellulose using a choline based ionic liquid analogue. Green Chem. 8, 784–786.

Abbot, A.P., Capper, G., McKenzie, K.J., Ryder, K.S. 2007. Electrodeposition of zinc–tin alloys from deep eutectic solvents based on choline chloride. J. Electroanal. Chem. 599, 288–294.

Abdelmouleh, M., Boufi, S., Salah, A.B., Belgacem, M.N., Gandini, A. 2002. Interaction of silane coupling agents with cellulose. Langmuir 18, 3203–3208.

Abdelmouleh, M., Boufi, S., Belgacem, M.N., Duarte, A.P., Salah, A.B., Gandini, A. 2004. Modification of cellulosic fibers with functionalised silanes: development of surface properties. Int. J. Adhes. Adhes. 24, 43–54.

Balazs, E.A. 1979. Ultrapure hyaluronic acid and the use thereof. US Patent 4,141,973.

Balazs, E.A. 2004. Viscoelastic properties of hyaluronan and its therapeutic use. In: Garg, H.G., Hales, C.A. (eds), Chemistry and Biology of Hyaluronan. Elsevier. Amsterdam, Netherlands. p 415.

Balazs, E.A., Denlinger, J.L. 1989. Clinical uses of HA. Ciba Foundation Symposium 143, 265–275.

Bell, T.W., Hou, Z., Luo, Y., Drew, M.G.B., Chapoteau, E., Czech, B.P., Kumar, A. 1995. Detection of creatinine by a designed receptor. Science 269, 671–673.

Block, S.S. 1991. Disinfection, Sterilization and Preservation. 4th ed. Lea & Febiger. Philadelphia, USA.

Bitter, T., Muir, H.M. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4, 330–333.

Bradford, M.M. 1976. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Brooks, G.F., Butel, J.S., Ornston, L.N. 1995. Medical Microbiology. 20th ed. Appleton & Lange. Connecticut, USA.

Brown, K.K., Ruiz, L.L.C., van de Rijn, I.., Greenne, N.D. 1994. Method for the microbiological production of non-antigenic hyaluronic acid. US Patent 5316926.

Brzoska, J.B., Shahidzzdeh, N., Rondelez, F. 1992. Evidence of a transition temperature for the optimum deposition of grafted monolayer coatings. Nature 360, 719–724.

Castellano, M., Gandini, A., Fabbri, P., Belgacem, M.N. 2003. Modification of cellulose fibres with organosilanes: Under what conditions does coupling occur? J. Colloid Interface Sci. 273, 505–511.

Chien, L.J., Lee, C.K. 2007a. Enhanced hyaluronic acid production in Bacillus subtilis by coexpressing bacterial hemoglobin. Biotechnol. Prog. 23, 1017–1022.

Chien, L.J., Lee, C.K. 2007b. Hyaluronic acid production by recombinant Lactococcus lactis. Appl. Microbiol. Biotechnol. 77, 339–346.

Chong, B.F. 2002. Improving the cellular economy of Streptococcus zooepidemicus through metabolic engineering. PhD Thesis, The University of Queensland.

Chong, B.F., Blank, L.M., Mclaughlin, R., Nielsen, L.K. 2005. Microbial hyaluronic acid production. Appl. Microbiol. Biotechnol. 66, 341–351.

Chung, C., Lee, M., Choe, E.K. 2004. Characterization of cotton fabric scouring by FT-IR ATR spectroscopy. Carbohydr. Polym. 58, 417–420.

Cleary, P.P., Larkin, A. 1979. Hyaluronic acid capsule: Strategy for oxygen resistance in group A Streptococci. J. Bacteriol. 140, 1090–1097.

Crater, D. L., Dougherty, B. A., van de Rijn, I. 1995. Molecular characterization of hasC from an operon for hyaluronic acid synthesis in group A Streptococci – demonstration of UDP-glucose pyrophosphorylase activity. J. Biol. Chem. 270, 28676–28680.

Cywes, C., Wessels, M.R. 2001. Group A Streptococcus tissue invasion by CD-44-mediated cell signaling. Nature 414, 648–652.

DeAngelis, P.L., 1999. Hyaluronic acid synthases: fascinating glycosyltransferases from vertebrates’ bacterial pathogens and algal viruses. Cell. Mol. Life Sci. 56, 670–682.

Della, V.F., Romeo, A. 1987. New polysaccharide esters and their salts. European Patent 216453.

Denyer, S. P. 1995. Mechanisms of action of antibacterial biocides. Int. Biodeterior. Biodegrad. 36:227–245.

Domingo, C., Loste, E., Fraile, J. 2006. Grafting of trialkoxysilane on the surface of nanoparticles by conventional wet alcoholic and supercritical carbon dioxide deposition methods. J. of Supercritical Fluids 37, 72–86.

Dougherty, B. A., van de Rijn, I. 1993. Molecular characterization of hasB from an operon for hyaluronic acid synthesis in group A Streptococci - demonstration of UDP-glucose dehydrogenase activity. J. Biol. Chem. 268, 169–175.

Dougherty, B. A., van de Rijn, I. 1994. Molecular characterization of hasA from an operon for hyaluronic acid synthesis in group A Streptococci. J. Biol. Chem.269, 169–175.

Edmonds, M.E., Foster, A.V. 2006. Diabetic foot ulcers. Brit. Med. J. 332, 407–410.

Ellwood, D.C., Evans, C.G., Dunn, G.M., McInnes, N., Yeo, R.G., Smith, K.J., Evans, C., Yeo, R., Evans, C.G.T. 1996. Production of hyaluronic acid. US Patent 5563051.

Esposito, E., Menegatti, E., Cortesi, R. 2005. Hyaluronan based microspheres as tools for drug delivery: A comparative study. Int. J. Pharm. 288, 35–49.

Fadeev, A.Y., MCarthy, T.J. 2000. Self-assembly is not the only reaction possible between alkyltrichlorosilanes and surfaces: monomolecular and oligomeric covalently attached layers of dichloro- and trichloroalkylsilanes on silicon. Langmuir 26, 7268–7274.

Franklin, T. J., Snow, G. A. 1981. Biochemistry of Antimicrobial Action. Chapman & Hall. London, UK. p 58.

Fouissac, E., Milas, M., Rinaudo, M. 1993. Shear rate, concentration, molecular weight and temperature viscosity dependences of hyaluronate, a wormlike polyelectrolytes. Macromolecules 26, 6945–6951.

Gatech, I., Popa, M., Rinaudo, M. 2005. Role of the pH on hyaluronan behavior in aquaeus solution. Biomacromolecules 6, 61–67.

Goh, L.T. 1998 Effect of culture conditions on rates of intrinsic hyaluronic acid production by Streptococcusequi subsp. zooepidemicus. PhD Thesis. University of Queensland.

Gottenbos, B., Grijpma, D.W., van der Mei, H.C., Feijen, J., Busscher, H.J. 2001. Antimicrobial effects of positively charged surfaces on adhering Gram-positive and Gram-negative bacteria. J. Antimicrob. Chemother. 48, 7–13.

Hardingham, T. 2004. Solution properties of hyaluronan. In: Garg, H.G., Hales, C.A. (eds), Chemistry and Biology of Hyaluronan. Elsevier. Amsterdam, Netherlands. p 6.

Hascall, V.C., Laurent, T.C., 1997, Hyaluronic acid: Structure and physical properties. Glycoforum – Hyaluronic acid Today. http://glycoforum.gr.jp/science/hyaluronan/HA01/HA01E.html (accessed May, 2008).

Hugo, W. B., and M. Frier. 1969. Mode of action of the antibacterial compound dequalinium acetate. Appl. Microbiol. 17, 118–127.

Jiang, S., Wang, L., Yu, H., Chen, Y., Shi, Q. 2005. Study on antibacterial behavior of insoluble quaternary ammonium. J. Appl. Polym. Sci. 99, 2389–2394.

Kim, J.H., Yoo, S.J., Oh, D.K., Kweon, Y.G. 1996. Selection of a Streptococcus equi mutant and optimization of culture conditions for the production of high molecular weight hyaluronic acid. Enzyme Microb. Technol. 19, 440–445.

Kirker, K.R.; Luo, Y.; Nielson, J.H.; Shelby, J.; Prestwich, G.D. 2002. Glycosaminogylcan hydrogel films as bio-interactive dressings for wound healing. Biomaterials 23,, 3661–3671.

Kogan, G., Šoltés, L., Stern, R., Gemeiner, P. 2007. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol. Lett. 29, 17–25.

Lazaridis, N.K., Kyzas, G.Z., Vassiliou, A.A., Bikiaris, D.N. 2007. Chitosan derivatives as biosorbents for basic dyes. Langmuir 23, 7634–7643.

Lapcik, L. Jr., Lapcik, L., Smedt, S.D., Demeester, J., Chabrecek, P. 1998. Hyaluronan: Preparation, structure, properties, and applications. Chem. Rev. 98, 2663–2684.

Leach, J.B., Schmidt, C.E. 2004. Hyaluronan. Encyclopedia of Biomaterials and Biomedical Engineering. Marcell Dekker. New York, USA. p 779–789.

LeGrange, J.D., Markham, J.L., Kurkjian, C.R. 1993. Effects of surface hydration on the deposition of silane monolayers on silica. Langmuir 9, 1749–1753.

Leonard, B.A., Woischnik, M., Podbielski, A. 1998. Production of stabilized virulence factor-negative variants by group A Streptococci during stationary phase. Infect. Immun. 66, 3841–3847.

Li, Y., Guang, H.M., Chung, T.S., Kulprathipanja, S. 2006. Effects of novel silane modification of zeolite surface on polymer chain rigidification and partial pore blockage in polyethersulfone (PES)–zeolite A mixed matrix membranes. J. Membr. Sci. 275, 17–28.

Liu, C.Y., Webster, D.A. 1974. A spectral characteristics and interconversions of the reduced of the reduced, oxidized, and oxygenated forms of purified cytochrome o. J. Biol. Chem. 249, 4261–4266.

Liu, J., Feng, X., Fryxell, G.E., Wang, L.Q., Kim, A.Y., Gong, M. 1998. Hybrid mesoporous materials with functionalized monolayers. Adv. Mater 10, 161–165.

Liu, L., Wang, M., Du, G., Chen, J. 2008. Enhanced hyaluronic acid production of Streptococcus zooepidemicus by an intermittent alkaline-stress strategy. Lett. Appl. Microbiol. 46, 383–388.

Liu, Y., Liu, Y.J. 2008. Biosorption isotherm, kinetics and thermodynamics. Sep. Purif. Technol. 61, 229–242.

Mao, Z., Chen, R.R. 2007. Recombinant synthesis hyaluronan by Agrobacterium sp. Biotechnol. Prog. 23, 1038–1042.

Mausolf, A., Jungmann, J., Robeneck, H., Phrem, P. 1990. Shedding of hyaluronate synthase from Streptococci. Biochem. J. 267, 191–196.

Mendichi, R., Schieroni, A.G. 2002. Fractionation and characterization of ultra-high molar mass hyaluronan: 2. On-line size exclusion chromatography methods. Polymer 43, 6115–6121.

Moseley, R., Walker, M., Waddington, R.J., Chen, W.Y.J. 2003. Comparison of the antioxidant properties of wound dressing materials—carboxymethylcellulose, hyaluronan benzyl ester and hyaluronan, towards polymorphonuclear leukocyte-derived reactive oxygen species. Biomaterials 24, 1549–1557.

Müller-Dethlefs, K., Hobza, P. 2000. Noncovalent interactions: a challenge for experiment and theory. Chem. Rev. 100, 143-167.

Narins, R.S., Brandt, F., Leyden, J., Lorenc, Z.P., Rubin, M., Smith, S. 2003. A randomized, double-blind, multicenter comparison of the efficacy and tolerability of Restylane versus Zyplast for the correction of nasolabial folds. Dermatol. Surg. 29, 588–595.

Nimrod, A., Greenman, B., Kanner, D. 1988. Method of producing high molecular weight sodium hyaluronate by fermentation of Streptococcus. US Patent 4,780,414.

Niaudet, B., Goze, A., Ehrlich, S.D. 1982. Insertional mutagenesis in Bacillus subtilis: Mechanism and use in gene cloning. Gene 19, 277–284.

O'Regan, M., Martini, I., Crescenzi, F., De Luca, C., and Lansing, M. 1994. Molecular mechanisms and genetics of HA biosynthesis. Int. J. Biol. Macromol. 16, 283–286.

Oh, S.Y., Yoo, D.I., Shin, Y., Kim, H.C., Kim, H.Y., Chung, Y.S., Park, W.H., Youk, J.H. 2005. Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydr. Res. 340, 2376–2391.

Ola, S.M.A.E., Kotek, R., White, W.C., Reeve, J.A., Hauser, P., Kim, J.H. 2004. Unusual polymerization of 3-(trimethoxysilyl)-propyldimethyloctadecyl ammonium chloride on PET substrates. Polymer 45, 3215–3225.

Parida, S.K., Dash, S., Patel, S., Mishra, B.K. 2006. Adsorption of organic molecules on silica surface. Adv. Colloid Interface Sci. 121, 77–110.

Prescott, A., Groton, M. 2002. Method for purifying high molecular weight hyaluronic acid. US Patent 6,660,853.

Puttick, M.P., Wade, J.P., Chalmers, A., Connell, D.G., Rangno, K.K. 1995. Acute local reactions after intraarticular hylan for osteoarthritis of the knee. J. Rheumatol. 22, 1311–1314.

Rangaswamy, V., Jain, D. 2008. An efficient process for production and purification of hyaluronic acid from Streptococcus equi subsp. zooepidemicus. Biotechnol. Lett. 30, 493–496.

Roy, D., Knapp, J.S., Guthrie, J.T., Perrier, S. 2008. Antibacterial cellulose fiber via RAFT surface graft polymerization. Biomacromolecules. 9, 9–12.

Salton, M. R. J. 1968. Lytic agents, cell permeability and monolayer penetrability. J. Gen. Physiol. 52, 277–252.

Schreiber, F. 2000. Structure and growth of self-assembling monolayers. Prog. Surf. Sci. 65, 151–256.

Scott, J.E. 1989. Secondary structures in HA solutions: Chemical and biological implications. The Biology of HA. Ciba Foundation Symposium 143, 6–14.

Stern, R., Asari, A.A., Sugahara, K.N. 2006. Hyaluronan fragments: an information-rich system. Eur. J. Cell. Biol. 85, 699–715.

Tashiro, T. 2001. Antibacterial and bacterium adsorbing macromolecules. Macromol. Mater. Eng. 286, 63–87.

van de Rijn, I., Kessler, R.E. 1990. Growth characteristics of group A streptococci in a new chemically defined medium. Infect. Immun. 27, 444–448.

Vasiliu, S., Popa, M., Rinaudo, M. 2005. Polyelectrolyte capsules made of two biocompatible natural polymers. Eur. Polym. J. 41, 923–932.

Walters, P.A., Abbott, E.A., Isquith, A.J. 1973. Algicidal activity of a surface-bonded organosilicon quaternary ammonium chloride. Appl. Microbiol. 25, 253–256.

Weigel, P.H., Hascall, V.C., Tammi, M. 1997. Hyaluronan synthases. J. Biol. Chem. 272, 13997–14000.

Weigel, P.H. 2004. Bacterial hyaluronan synthases. Glycoforum – Hyaluronic acid Today. http://glycoforum.gr.jp/science/hyaluronan/HA06/HA06E.html (accessed May, 2008).

Weissman, B., Meyer, K. 1954. The structure of hyalobiuronic acid and of hyaluronic acid from umbilical cord. J. Am. Chem. Soc. 76, 1753–1757.

Wessels, M.R., Moses, A., Goldberg, J.B., DiCesare, T.J. 1991. Hyaluronic acid capsule is a virulence factor for mucoid group A Streptococci. Proc. Natl. Acad. Sci. USA 88, 8317–8321.

Westphal, G., Hahn, M., Lippert, E., Hoffmann, K., Kaukel, P. 2002. Isolation of hyaluronic acid, e.g. from fermentation solutions, comprises precipitation with an ethoxylated fatty amine hydrochloride, useful in medical, pharmaceutical and cosmetic applications, DE Patent 10019868 A1.

Wibowo, N., Setiyadhi, L., Wibowo, D., Setiawan, J., Ismadji, S. 2007. Adsorption of benzene and toluene from aqueous solutions onto activated carbon and its acid and heat treated forms: influence of surface chemistry on adsorption, J. Hazard. Mater. 146, 237–242.

Widner, B., Behr, R., Von Dollen, S., Tang, M., Heu, T., Sloma, A., Sternberg, D., DeAngelis, P.L., Weigel, P.H., Brown, S. 2005. Hyaluronic acid production in Bacillus subtilis. Appl. Environ. Microbiol. 71, 3747–3752.

Yamada, T., Kawasaki, T. 2005. Microbial synthesis of Hyaluronic acid and Chitin: New approaches. J. Biosci. Bioeng. 99, 521–528.

Yang, P.F., Lee, C.K. 2007. Hyaluronic acid interaction with chitosan–conjugated magnetite particles and its purification. Biochem. Eng. J. 33, 284–289.

Yoshida, W., Castro, R.P., Jou, J.D., Cohen, Y. 2001. Multilayer alkoxysilane silylation of oxide surfaces. Langmuir 17, 5882–5888.

Yu, H., Stephanopolous, G. 2008. Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid. Metabol. Eng. 10, 24–32.

Yun, Y.H., Goetz, D.J., Yellen, P., Chen, W. 2004. Hyaluronan microspheres for sustained gene delivery and site specific targeting. Biomaterials 25, 147–157.

Xu, H., Ito, T., Tawada, A., Maeda, H., Yamanokuchi, H., Isahara, K., Yoshida, K., Uchiyama, Y., Asari, A. 2002. Effect of hyaluronan oligosaccharides on the expression of heat shock protein 72. J. Biol. Chem. 277, 17308–17314.

Zhou, H., Ni, J., Huang, W., Zhang, J. 2006. Separation of hyaluronic acid from fermentation broth by tangential flow microfiltration and ultrafiltration. Sep. Purif. Technol. 52, 29–38.