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- Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

1. Background

Alginates form an important family of biopolymers of both technological and scientific interest. These polymers are linear polysaccharides, which are composed of variable amounts of (1–4)-β-D-mannuronic acid and its epimer, α-L-guluronic acid. Alginates present a wide range of applications, acting for example as stabilizing, thickening, gel or film-forming agents, in various industrial fields. Currently, commercial alginates are extracted from marine brown algae and are used for a variety of applications, mainly in the food and pharmaceutical industries [1]. Increasingly new applications are being discovered for these polymers; an example of this is its use as a source of soluble fiber [2].

Alginates extracted from algae are relatively cheap products, having selling prices in the range US$ 5–20/kg for the majority of the applications [1]; however, alginates of very high purity are used in the pharmaceutical field and these are sold for up to US$ 40,000/kg [1]. The algal alginates have several problems concerning their production which may limit their use in many interesting contexts, especially in the pharmaceutical and chemical industries, where polymers with a very well defined composition, are required. Algal alginates are complex mixtures of polymers, exhibiting a wide range of molecular masses and compositions (G-M) and blocks distribution. These characteristics are practically impossible to control using the current procedure, which relies on harvesting algae from the ocean where there is no control over the environmental conditions, which in turn define the molecular characteristics of the polymer.

Alginates are also produced by bacteria and many of their physicochemical characteristics are similar to those of algae, so that they can be used for the same applications as algal alginates, as well as in other more sophisticated contexts. Alginates produced by microorganisms differ from those of algae because bacterial polymers are acetylated [3]. In addition, bacterial alginates usually have a higher molecular mass than the algal polymers (ranging from 48 to 186 kDa). A molecular mass as high as 4,000 kDa for the polymer synthesized by a mutant strain of A. vinelandii has been reported [4]. Both acetylation and molecular mass directly affect directly the viscosity and other rheological properties of alginate solutions and, therefore, this would determine its utility in specific applications of alginate in the food and pharmaceutical fields.

In the bacterial world, alginates are produced by Pseudomonas and Azotobacter species. A considerable amount of work has been published regarding the production of alginate by the Pseudomonas species, an interest driven mainly by the fact that alginate plays an important role in the pathogenicity of Pseudomonas aeruginosa in cystic fibrosis. In contrast to P. aeruginosa, A. vinelandii is a non-pathogenic soil bacterium, which can be used for the development of biotechnological process to produce alginate. This characteristic, as well as the interest in the role that alginate plays in cyst formation has motivated the study of various aspects concerning the production of alginate from Azotobacter. Furthermore, the availability of the complete sequence of A. vinelandii genome has made this bacterium an ideal model of study, from both technological and scientific points of view.

A. vinelandii has another interesting characteristic: under unbalanced growth conditions, this bacterium produces poly-β-hydroxybutyrate (PHB), a polymer of the polyhydroxyalcanoates (PHAs) family of polyesters, which are synthesized by a wide range of bacterial and archaeal species to form carbon and energy reserve materials [5]. PHAs are present in the cytoplasm of bacterial cells as water insoluble granules. Besides playing an important role as a reserve polymer, PHB has been implicated in supporting nitrogen fixation [6]. In A. vinelandii, PHB is related to the differentiation process this bacterium undergoes in order to produce cysts resistant to desiccation, as numerous granules are present in mature cysts. However, under laboratory conditions, mutants impaired in PHB synthesis formed mature cysts, resistant to desiccation [7].

PHAs have been drawing attention because they are biodegradable and biocompatible thermoplastics, which can be processed to create a wide variety of consumer products, including plastics, films, and fibers. Imperial Chemical Industries (ICI) started the industrial production of these polyesters in 1982 with the trade name of "Biopol" as a biodegradable substitute for some petroleum-derived plastics [8]. Nowadays, Metabolix and the Kaneka Corporation are producing industrial PHAs [5].

Subjects covered by this review include; research concerning the production of alginate and PHB by A. vinelandii, particularly aspects which include the molecular regulation of the production of the two polymers, the construction of recombinant strains for producing more or higher quality alginate and, or PHB, the fermentation conditions which result in attractive bioprocess yields and the potential for scaling-up such processes,

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