Increases in alginate production and transcription levels of alginate lyase (alyA1) by control of the oxygen transfer rate in Azotobacter vinelandii cultures under diazotrophic conditions

BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

Respiration in Azotobacter vinelandii and its relationship with the synthesis of biopolymers

Azotobacter vinelandii is a gram-negative soil bacterium that produces two biopolymers of biotechnological interest, alginate and poly(3-hydroxybutyrate), and it has been widely studied because of its capability to fix nitrogen even in the presence of oxygen. This bacterium is characterized by its high respiration rates, which are almost 10-fold higher than those of Escherichia coli and are a disadvantage for fermentation processes. On the other hand, several works have demonstrated that adequate control of the oxygen supply in A. vinelandii cultivations determines the yields and physicochemical characteristics of alginate and poly(3-hydroxybutyrate). Here, we summarize a review of the characteristics of A. vinelandii related to its respiration systems, as well as some of the most important findings on the oxygen consumption rates as a function of the cultivation parameters and biopolymer production.

Simultaneous environmental manipulations in semi-perfusion cultures of CHO cells producing rh-tPA

We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31ºC) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32 percent at 34ºC and 55 percent at 31ºC, compared to cultures at 37ºC. Similar behaviour was observed in cultures at 34ºC using galactose as a carbon source. Nonetheless, at 31ºC, the specific productivity of rh-tPA strongly decreased (about 58 percent) compared to the culture at 37ºC. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34ºC. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112 percent over that obtained in semi-perfusion culture carried out at 37ºC with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.