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Fermentation strategies for the PHB production using A. vinelandii
- Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

7. Fermentation strategies for the PHB production using A. vinelandii

A. vinelandii usually produces PHB. This polymer forms highly crystalline solids, resulting in the production of brittle plastics [8]. However, by using a fermentation strategy consisting in the addition of valerate, heptanoate or nonanoate to a culture of A. vinelandii UWD grown in glucose, Page et al. [80] were able to obtain the copolymer poly(Hydroxybutyrate-Co-Hydroxyvalerate) (P(HB-co-HV)), which produces plastics exhibiting better mechanical properties. These authors reported β-hydroxyvalerate contents ranging from 8.5 to 52 mol %, depending on the concentration of valerate used.

Another aspect which has been evaluated, regarding the quality of PHAs produced by A. vinelandii refers to the degree of polymerization. This characteristic is expected to affect the mechanical properties of the plastics produced [81]. In the A. vinelandii strainUWD the formation of a very high molecular weight PHB (4 million Daltons) is promoted by some of the non-sugar components of beet molasses. In fact, the molecular weight of PHB can be altered between one and four million Daltons depending on the nature of the carbon source used [81].

A wider use of PHA-derived plastics has been hampered because of their high production costs [82]. The cost of the carbon source contributes significantly to the overall production cost of PHAs [82]. Because of their low price, unpurified organic wastes from agriculture and food processing can be excellent substrates for the bacteria. On this regard, Page [58], reported that the A. vinelandii strain UWD is able to grow and produce PHB up to 2.5 g/L using glucose and it can also produce the polymer from fructose, sucrose, maltose, gluconate or glycerol as carbon sources. However, unrefined carbon sources such as corn syrup, cane molasses, beet molasses, or malt extract, also support PHB formation, obtaining yields of PHB comparable to, even better than the refined sugars. Beet molasses and malt extract promoted higher polymer production per liter (2.74 and 2.80 g/L respectively) due to a growth stimulatory effect [58]. The addition of valerate in a fed-batch fermentation using beet molasses as carbon source, sustained the production of (P(HB-co-HV) with a 20 % of hydroxyvalerate, demonstrating that the production of copolymers is also feasible using unrefined substrates [83]. It has been reported that PHA production can be increased and the yield improved, by supplementing a small amount of complex nitrogen sources [80]. The supplementation with fish peptone, proteose peptone, and yeast extract promoted a significant increase in the production of PHB per liter (up to 7.5 g/L). An alternative approach which consisted of supplementing fish peptone to a fed-batch culture, resulted in a high PHB concentration of 32 g/L after 47 h of culture [84].

Another factor influencing production costs is the recovery of the product. Page and Cornish [84] reported that A. vinelandii strain UWD cells, when cultured in medium supplemented with fish peptone, become fragile and break easily. Therefore, a simple treatment with 1 M NH4OH allows the separation of a highly pure PHA. This phenotype has permitted the development of an economical recovery method [84]. Using a two stage fermentation process as a strategy for improving the production of PHAs. Chen and Page [85] increased the concentration of the polymer produced by up to 36 g/L (in a 2.5 liter fermentor) and also notably improved productivity, by up to 1.05 g/L * h. This process was designed using aeration to promote growth and to suppress PHB production in the first phase, while lower aeration of a culture containing fish peptone as a nitrogen source was used to promote PHB formation in the second phase, taking advantage of the higher biomass achieved.

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