Hydrogels and Biodegradable Polymers for ... - ACS Publications

Chapter 18

Microbes in Polymer Chemistry V. Crescenzi and M. Dentini

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 20, 2016 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0627.ch018

Department of Chemistry, University La Sapienza, P. le A. Moro 5, 00185 Rome, Italy

A few examples of the impact that conventional and advanced microbial technologies already have and may, eventually, have on polymer chemistry are outlined. From microbial biomass in addition to enzymes (not considered in this review), a variety of polymeric materials of potential commercial importance can be obtained including carbohydrate polymers, aliphatic polyesters and polyaminoacids. In addition, a number of monomelic compounds of interest in polymer chemistry for the synthesis of industrially important macromolecules (e.g. polyamides and polyesters) can be obtained via fermentation of wild, mutated and/or genetically engineered microbial strains. A better exploitation of natural, renewable resources, e.g. microbial biomass, for the production of a larger number of bifunctional species amenable to polymerization will also help increase the ecological friendliness of raw materials industries.

Microorganisms have long been utilized to produce, in some cases at the industrial level, a variety of organic compounds, in particular high added-value biologically active species. This has promoted the development of conventional and advanced biotechnologies. In addition, the many possibilities offered to the "polymer community" by a wise exploitation of microbial biomass is not yet fully recognized. Indeed, selected microbial cultures are, or may become, valuable sources of important classes of polymers and of a number of monomers or reactive intermediates. Microbial polymers include, as is well known, enzymes, polysaccharides, polyesters, and polyaminoacids: some of these natural macromolecules are presently considered as irreplaceable commodities or specialties for a number of applications. Monomers most conveniently derived from microbial fermentation include L-lactic acid and acrylonitrile, but the list is likely to widen 0097-6156/96/0627-0221$15.00/0 © 1996 American Chemical Society

In Hydrogels and Biodegradable Polymers for Bioapplications; Ottenbrite, Raphael M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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considerably in the near future: this should be significant for the ecologically oriented chemical industry. There follows a few specific examples of the impact of microbial technologies on polymer chemistry. For brevity, die presentation is limited to two cases (with which the authors have direct working experience), namely : I). - industrial microbial polysaccharides ; Π). exocellular polyaminoacids. In addition, the case of monomers from microbial biomass - a topic not yet reviewed, to the authors' knowledge - is presented in a schematic way including some actual, stimulating examples. I) - Microbial Polysaccharides. - Some comments on "where we are". A large number of exocellular and capsular polysaccharides are known and new examples are being discovered and described in the scientific literature at a steadily increasing pace. Limiting attention to examples of actual interest for the polymer industry, a list of polysaccharides already commercialized and obtainable exclusively - with the exception of hyaluronic acid and cellulose - from microbial fermentations is given in Table I. Most prominent application of each species is briefly indicated in the foot note to Table I. Detailed discussions on this topic can be found in recent reviews (1-3): suffice here to mention that for such end uses, the polysaccharides listed in Table I are considered rather unique and, often, irreplaceable. They include practically all desirable properties for technological biopolymers. A number of studies on the solution and gelling properties of these microbial polysaccharides and their derivatives have been carried out in the last twp decades in our laboratory. In particular, polysaccharides studied include: xanthan (4-6), the "gellan family" polysaccharides (7-10), succinoglycan (11-12), and scleroglucan (1314). One important feature shared by these biopolymers, which attracts particular attention, is their ability to adopt ordered stable conformations even in very dilute aqueous solutions. These conformations, with the notable exception of succinoglycan, are double (the "gellan family" polysaccharides) or triple stranded (scleroglucan). The main aim of our work, like that of many other researchers, has been to contribute to the understanding of how chemical structure determines the adoption of ordered conformation, and how the latter controls the formation of three dimensional networks (physical gels).


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Table I. Industrial Microbial Polysaccharides


biopolymer

main microbial source

typical applications

Polyelectrolytes hyaluronan

a

Streptococcus spp. Xanthomonas campestris

c, d, e

gellan

Aureomonas elodea

d,f

welan

Alcaligenes spp.

i,g

Pseudomonas spp.

i>g

xanthan

succinoglycan Non-ionic cellulose

Acetobacter xylinum

curdlan

Agrobacter. spp.

pullulan dextran scleroglucan

a f,d h

Aureobasidium pullulans Leuconostoc spp.

a, b

Sclerotium spp.

a = pharmaceuticals ; b = chemical gels ; c = oil recovery ; d = food additives ; e = textiles ; f = physical gels ; g = drilling fluids; h = films ; i = rheology modifier.



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Clearly, a detailed knowledge of the characteristic conformational ordering of stereoregular microbial polysaccharide chains is fundamental to obtaining a deeper insight of the molecular basis of their novel macroscopic solution properties, especially Theological and gelation. This, in turn, permits a better industrial exploitation of these biopolymers. In addition, in view of the biocompatibility of these polysaccharides we have also prepared novel derivatives with the purpose of 1) studying the influence of chemical functionalization on the stability of ordered conformations and hence gelling ability (e.g. benzyl esters of gellan of different degrees of esterification (15-16); 2) preparing and characterizing novel biomaterials - including soluble derivatives and cross-linked hydrogels - potentially suitable for sustained drug release formulations (17). In the latter context, we recently succeeded in preparing a variety of chemical hydrogels built up by gellan chains engaged at random through their carboxylate functions in ester formation with simple difunctional reagents (18). The materials obtained exhibit interesting swelling properties when in contact with aqueous salt media, as shown in Fig. 1. They appear to have potential as controlled release agents for selected cationic drugs, as shown by the preliminary, in vitro results outlined in Fig. 2. While continued basic research on this topic has obvious relevance, it could be claimed that applied research is no longer necessary. We consider that this is not so provided that some practical guidelines are observed. In this context, we wish to submit the following schematic considerations which, for a comprehensive overview of the topic, should be considered in conjunction with other reviews (2,3). a) - In some cases, the performance/cost ratio of microbial polysaccharides appears questionable for certain end uses. An approach to reducing polysaccharides production costs and, at the same time, alleviating specific pollution problems consists of the "target oriented" search, identification, and use of strains - good producers of polysaccharides - living on waste materials. In this regard at least two recent, interesting examples can be mentioned dealing with the production of polysaccharides - displaying good rheological properties - by different bacterial strains fed either with whey permeate (19) or with maple sap (20). If such bacterial strains can also be proved "beyond all doubts" from the biocompatibility standpoint - as, for instance, is obviously the case for Lactobacteriaceae (21) - the polysaccharides produced should



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Figure 1. Swelling equilibria of a gellan cross-linked hydrogel at different ionic strengths (NaCl, 25°C). The sample was prepared following the procedure given elsewhere (6) for the synthesis of simple, linear alkyl and arylalkyl esters of gellan but using α,ω-dibromo octane as reticulating agent. Estimated degree of cross-linking: 0.5.

Figure 2. Time course of the release of Chlorohexidine from the cross-linked gellan hydrogel of Fig. 1. (t=0 conditions: 4mg of drug/70mg of hydrogel; 0.1 M NaCl, 25°C).


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find easier approval for food uses by relevant authorities (thus cutting R&D costs and gaining in commercial value). b) - A further development of what today may be called "microbial polysaccharide science and technology" - an interdisciplinary field encompassing expertises such as microorganism genetics and physiology, carbohydrate chemistry and biochemistry, and polymer chemistry - will certainly result from a more extensive use of advanced biotechnologies. This may allow the enzymatic synthesis in cell free systems of valuable biopolymers (e.g. very pure, very high molecular weight hyaluronic acid (22)) and the production of tailor-made microbial polysaccharides for specific biomedical or food applications where parameters such as production costs/yields may become only marginally relevant. c) - The understanding of the fine-tuned relationships between structure and properties - in the solid state, in solution, and in the gel state - of the often structurally complex microbial polysaccharides is still being developed. This understanding is essential for a better exploitation of polysaccharides already known, for the selection among polysaccharides being discovered of those better suited for industrial R&D, and for an intelligent guide to the synthesis of novel polysaccharide-based derivatives. The latter include chemically functionalized polymeric materials with potential in biomedicine (e.g. as pro-drugs) and highly hydrophylic chemical gels target oriented primarely to controlled drug-delivery formulations. Π) Microbial Polyaminoacids. - Biopolymers "in search of applications" (23). In the early twenties, a milestone in understanding the structure and the biological significance of carbohydrate polymers from pathogenic bacteria was represented by the discovery that serological, type specific antigens of bacteria of the genus Streptococcus are polysaccharides (24). This opened the way to important progress in the use of such polysaccharides as immunogens, a subject of actual studies on the formulation of human vaccines. In 1925, Lemoigne, a microbiologist at the Pasteur Institute in Paris, first described poly(3-hydroxybutyrate) as an endocellular polyester obtainable from bacterial cultures (25).



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These were indeed "roaring" years for microbial polymers. In fact, between 1921 and 1937 high molecular weight polyglutamates of microbial origin were also identified, isolated and partially characterized (26). Ironically, a fate common to the three types of biopolymers mentioned above - with the possible exception of polysaccharides - is that, after their discovery, many years had to elapse before they fully deserved in polymer chemistry. Indeed, in the case of polyglutamates (more in general of microbial polyaminoacids) one can still speak of "biopolymers in search of application" (23). At present, polyglutamates (which consist of glutamic acid units linked between the α-amino and the γ-carboxylic acid functionalities and hence are named poly-y-glutamates (γ-PGA)) of different stereoregularity (i.e. D- and L-glutamate content) and molecular weight (normally exceeding 1θ5 ) are produced exocellularly in very good yields from species of the genus Bacillus. Recently, a revival in interest in these biopolymers has lead to a number of publications dealing in particular with the biosynthesis, the resulting chains stereochemistry, and with the production of derivatives with potential as biomaterials (23, 27). In a companion paper (28), we present original data on the physico-chemical characterization of γ-D-PGA (approximately 96 % D) in dilute aqueous solution. In our laboratory, work is in progress also on the synthesis of novel γ-PGA based hydrogels. In the context of this review, it should be underlined that γ-PGAs can be easily obtained by fermentation of selected Bacillus licheniformis strains with yields of tens of g/L. They are water soluble polyelectrolytes capable of interacting specifically with divalent counterions (without precipitation) and tervalent counterions (with gelation/precipitation). They exhibit novel conformation-dependent solution properties (28), and lend themselves to derivatization leading to organic solvent soluble alkyl and arylalkyl polyesters (23, 27) for example, and to cross-linked, highly water swellable hydrogels (18). Finally, it is worth mentioning that from the culture broths of Streptomyces albulus, low molecular weight ε-poly-L-lysine can be extracted (29). This unusual polyaminoacid, whose properties have been studied so far in a preliminary fashion, but sufficient to characterize ε-poly-L-lysine chains as conformationally adaptable and as efficient séquestrant of dye molecules and divalent ions (30), deserves more attention from polymer chemists.


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ΠΙ) Examples of Monomers from Microbial Sources.


- An ecological future for the polymer industry. An incomplete list of monomelic compounds of importance for the synthesis of a variety of macromolecules and which are produced, or may be produced in a probably near future, using microbial biomass is given in Table Π. Some of these monomelic compounds are now discussed. In polymer chemistry, lactic acid is an important feedstock for the production of biodegradable lactide polymers which are especially attractive for biomedical applications. A number of publications have recently appeared on the fermentative production of L-lactic acid in which attention is focused on yields and product recovery. For example, an innovative system for lactic acid recovery by ion-exchange resins has been proposed (31): the final yield obtained using Lactobacillus casei subsp. casei (glucose as main nutrient) is quite high and the resulting monomer (exclusively the L form) of purity adequate ( > 99 %) for its use in the synthesis of polylactides. Acrylamide can be produced industrially in a convenient fashion by selective hydration of acrylonitrile using as catalyst Rhodococcus rhodocrous Jl nitrile hydratase. According to a recent report (19), after fermentation for 72 h at around 20 °C with a cell yield of 28 g/L, the acrylamide productivity is higher than 7000 g/g cells. The final concentration of acrylamide is 40 % maintaining the acrylonitrile concentration at 6 %. Nitto Chemical Industry Co. of Japan claims to produce acrylamide at kiloton scale using R. rhodochrous Jl fermentation (32). Concerning 1,3-propanediol, a fermentative production from glycerol using Clostridium butyricum , up to a scale of 2 m3, has been reported (33). Selected strains of C. butyricum are able to convert up to 110 g/L of glycerol to 56 g/L of 1,3-propanediol in 29 h. These results are quite interesting in the context of the present review for the following two main reasons. Firstly, glycerol is available in large amounts at low price mainly as a by-product of the detergent industry. Secondly, 1,3-propanediol is a monomer suitable for the synthesis of a variety of polyesters, in particular for light-stable and/or biocompatible plastics. In addition, it should be pointed out that conventionally produced 1,3-propanediol has not gained economic and technological importance mainly because its synthesis is carried out by hydrolysis of acrolein and subsequent catalytic hydrogénation with relatively low yields ( about 43 % (21)). 2,3-Butanediol is an interesting chemical which can find a



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Table II. Industrially relevant monomers which are produced or may be produced from microbial sources

monomer (substrate) L(+)-lactic acid (carbohydr.) acrylamide (acrylonitrile)

microorg. (recent example) Lactobacillus casei. Rhodococcus rhodocrous Jl

1,3-propanediol (glycerol)

Clostridium butyricum

2,3-butanediol (carbohydr.)

Enterobacter aerogenes

stage

industrial (70% of total) industrial pilot research research

fumaric acid (carbohydr.)

Rhizopus arrhizus

2,3 and 3,4-cis dihydrodiols (benzene & deriv.)

Pseudomonas spp

research

engineered E. coli

research

adipic acid (glucose)



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number of applications, in addition to polymer synthesis. Recent reports suggest that for this diol also microbial production (e.g. using Enterobacter aerogenes strains) may soon become competitive with chemical synthesis (34). The development of a really competitive microbial production of fumaric acid has proved more difficult. Fumaric acid is conventionally prepared by acid isomerization of maleic acid, a bulk chemical commodity. Nevertheless, it is of interest at least to mention that this dicarboxylic monomer can be obtained from carefully controlled fermentation of Rhizopus arrhizus at a final concentration of 47 g/L (5 days, 47 % yield (35)). Passing to the "cis-diol" type compounds, it should be recalled that the biosynthesis of 5,6-dihydroxycyclohexa-l,3-diene by means of the bacterial oxidation of benzene using genetically manipulated Pseudomonas putida strains has been reported in 1970 (36). Simple ester derivatives of this compound are readily polymerized, using radical initiators, to give high molecular weight polymers which are soluble in common organic solvents. Moreover, such polymers undergo smooth conversion, on heating at 140-240 °C, into polyphenylene a polymer of interest for its thermal stability and electrical conductivity (when doped), but obtainable only in oligomeric form via conventional chemical routes. P. putida strains are an interesting example of the intelligent search for microorganisms living on waste materials (see also Section I). In this case, environmentally dangerous chemical wastes are used, which can produce monomelic compounds not otherwise obtainable in comparable yields and steric purity. More recently, it has been reported (37) that it is now possible to obtain 2,3- and 3,4-cis-dihydrodiols of either enantiomeric form in a predictable manner by the action of P. putida and E. coli engineered strains on carefully selected substrates (i.e. monosubstituted benzenes) and hydrogenolysis of ensuing metabolites. Such dihydrodiols should prove useful chiral syntons in organic synthesis as well as in polymer synthesis. Of similar keen interest are recent reports (38) indicating that lactones and lactames related to carbohydrates can be obtained by the functionalization of 1 -chloro-2,3-dihydroxycyclohexa-4,6-diene biocatalytically generated by fermentation of cholorobenzene with P. putida 39D. This section concludes with what we consider a real "up-beat note" by making reference to a recent paper by Draths and Frost (39). These authors have addressed with success on a laboratory scale the problem of the biocatalytic conversion of D-glucose into cis,cis-



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muconic acid (using heavily genetically engineered E. coli strains) and subsequent catalytic hydrogénation to afford adipic acid. The possibility of obtaining in the future adipic acid, primarely used in production of nylon-6,6 and whose annual global demand is of the order of billion Kg, following the route schematically outlined above (thus, eventually, reducing at least partially the use of conventional, highly polluting chemical processes) is very exciting and probably one of the most convincing examples of the impact microbial technologies may have on polymer chemistry and technology.

Acknowledgements. The A.A. wish to acknowledge the financial support of the Italian National Research Council, Progetto Finalizzato Chimica Π, Rome. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Sutherland, I. W.; Biotechnology of Microbial Polysaccharides, Cambridge Studies on Biotecnology - 9, Cambridge University Press, 1990. Crescenzi, Y.;Trends in Polymer Science 1994, 2, 104. Crescenzi, V.; Biotechnol. Progress, in press. Dentini, M.; Crescenzi, V.; Blasi, D.; Int. J. Biol. Macromol. 1984,6 (2), 93. Coviello, T.; Kajiwara, K.; Burchard,W.; Dentini, M . ; Crescenzi, V.; Macromolecules 1986, 19, 2826. Coviello, T.; Burchard, W.; Dentini, M.; Crescenzi, V.; Macromolecules 1987, 20, 1102. Crescenzi,V.; Dentini, M.; Dea, I.C.M.; Carbohydr. Res. 1987,160, 283. M . Dentini; T. Coviello; W. Burchard; V. Crescenzi; Macromolecules 1988, 21, 3312. Crescenzi,V.; Dentini, M.; Coviello, T.; in: Novel Biodegradable Microbial Polymers. E. A. Dawes Ed., NATO ASI Series E: Applied Sciences, Vol. 186, Kluwer Academic Pubis., Dordrecht, Netherlands, 1990, pp 277-284. Crescenzi, V.; Dentini, M.; Coviello, T.; in: Frontiers in Carbohydrate Research 2., R.Chandrasekaram, Ed., Elsevier, New York, 1991, pp 100114. Dentini, M.; Crescenzi, V.; Fidanza, M.; Coviello, T.; Macromolecules, 1989, 22, 954. Fidanza, M . ; Crescenzi,V.; Dentini, M . ; Del Vecchio, P.; Int. J. Biol. Macromol. 1989, 11, 372. Crescenzi, V.; Gamini, Α.; Rizzo, R.; Meille, S.V.; Carbohydrate Polymers 1988, 9, 169. Coviello,T.; Dentini, M.; Crescenzi, V.; Vincenti, Α.; Carbohydrate Polymers 1995, 26, 5.. Crescenzi, V.; Dentini, M . ; Segatori, M . ; Tiblandi, C.; Callegaro, L.; Benedetti, L.; Carbohydrate Research 1992, 231, 73. Crescenzi, V.; Dentini, M.; Maschio, S.; Segatori, M.; Makromol. Chem., Makromol. Symp.,1993, 76, 95.


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17. 18. 19. 20.


21. 22. 23.

24. 25. 26. 27.

28. 29. 30. 31. 32. 33. 34. 35. 36.

37. 38. 39.

Sanzgiri, Y.D.; Maschio, S.; Crescenzi, V.; Callegaro, L.; Topp, E.M.; Stella, V.J.; J. Controlled Release 1993, 26, 195. Crescenzi, V.; Dentini, M.; in preparation. Flatt, J.H.; Hardin, S.H.; Gonzalez, J. M.; Dogger, D. E.; Lightfoot, Ε. N.; Cameron, D. C.; Biotechnol. Progress 1992, 8, 327. Morin, Α.; Moresoli, C.; Rodrigue, Ν.; Dumont, J.; Racine, M.; Poitras E.; Enzyme Microbiol. Technol. 1993, 15, 500. Gruter, M.; Leeflang, B. R.; Kuiper, J.; Kamerling J. P.; Vliegenthart, F. G.; Carbohydr. Res. 1993, 239, 209. Dougherty, B.A.; van de Rijn, I.; J. Biol. Chem. 1993, 268, 7118. Gross, R. Α.; Birrer, G. Α.; Cromwick, A.-M.; Giannos, S. Α.; McCarthy, S. P.; Biotechnological Polymers: Medical, Pharmaceutical, and Industrial Applications, Gebelein, C. G., ed., Technomic Publ. Co., 1993, Part ΙΠ, pp 200-214. Heidelberger, M.; J. Am. Chem. Soc. 1993, 115, 2558. Lemoigne, M.; Ann. Inst. Pasteur (Paris), 1925, 39, 144. references in: Nitecki, D. E.; Goodman, J. W.; Chemistry and Biochemistry of Aminoacids, Peptides and Proteins, Weinstein, B., ed., Marcel Dekker, 1971, Vol. I, pp 87-126. - a) - Giannos, S. Α.; Shah, D.; Gross, R. Α.; Kaplan, D. L.; Mayer, J. M.; in: Novel Biodegradable Microbial Polymers., Dawes, Ε. Α., ed., Series Ε, Applied Sciences, Vol. 186, Kluwer Academic Publ.,1990, pp 457-460. - b) - Borbély, M.; Nagasaki, Y.; Borbély, J.; Fan, Κ.; Bhogie, Α.; Sevolan, Μ.; Polymer Bulletin, 1994, 32, 127. Crescenzi, V.; D'Alagni, M.; Dentini, M.; Segre, A.-L.; Ferretti, J. Α.; Biorelated Polymers; Am. Chem. Soc. Symposium Series, Am. Chem. Soc., Washington, D.C. in press. Shima, S.; Fukuhara, Y.; Sakai, H.; Agric. Biol. Chem. 1982, 46, 1917. Takagishi, T.; Kuroki, N.; Shima, S.; Sakai, H.; Yamamoto, H.; Nakazawa, Α.; J. Polymer Sci., Polymer Letters Ed. 1986, 23, 1917. Vaccari, G.; Gonzalez-Vara y R., Α.; Campi, A. L.; Dossi, E.; Brigidi, P.; Matteuzzi, D.; Appl. Microbiol. Biotechnol. 1993, 40, 23. Nagasawa, T. Shimizu, H., Yamada, H., Appl. Microbiol. Biotechnol., 1993, 40, 189. Gunzel, B.; Yonsel, S.; Deckwer, W.-D.; Appl. Microbiol. Biotechnol.. 1991, 36, 289. Zeng, A.-P; Biebl, H.; Deckwer, W.-D.; Appl. Microbiol. Biotechnol. 1990, 33, 264. Moresi, M.; J. Chem. Technol. Biotechnol. 1992, 54, 283. - a) - Gibson, D. T.; Cardini, G. E.; Maseles, F. C.; Kalio, R. E.; Biochemistry 1970, 9, 1631. - b) - Ballard, D. G. H.; Courtis, Α.; Shirley, I. M.; Taylor, S. C.;J.Chem. Soc., Chem. Comm. 1983, 954. Boyd, D. R.; Sharma, N. D.; Barr, S. Α.; Dalton, H.; Chima, J.; Whited, G.; Semayer, R.; J. Am. Chem. Soc. 1994, 116, 1147. Hudlicki, T.; Rouden, J.; Luna, H.; Allen, S.; J. Am. Chem. Soc. 1994, 116, 5099. Draths, Κ. M.; Frost, J. W.; J. Am. Chem. Soc. 1994, 116, 399.

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