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Aims: The sigma E (AlgU) in Azotobacter vinelandii has been shown to control the expression of cydR gene, a repressor of genes of the alternative respiratory chain, and alginate has been considered a barrier for oxygen diffusion. Therefore, the aim of the present study was to compare the respiratory activity of an alginate nonproducing strain, lacking the sigma factor E (algU-), and polymer-producing strains (algU+) of A. vinelandii under diazotrophic conditions at different aeration conditions., Methods and Results: Our results reveal that under diazotrophic and high aeration conditions, A. vinelandii strain OP (algU-) had a specific oxygen consumption rate higher (30 and 54%) than those observed in the OP algU+-complemented strain, named OPAlgU+, and the ATCC 9046 respectively. However, the specific growth rate and biomass yields (based on oxygen and sucrose) were lower for OP cultivations as compared to the algU+ strains. These differences were partially explained by an increase in 1·5-fold of cydA relative expression in the OP strain, as compared to that obtained in the isogenic OPAlgU+ strain., Conclusions: Overall, our results confirm the important role of algU gene on the regulation of respiratory metabolism under diazotrophic growth when A. vinelandii is exposed to high aeration., Significance and Impact of the Study: This study highlights the role of AlgU to control respiration of A. vinelandii when exposed to diazotrophy., (© 2018 The Society for Applied Microbiology.)

Alginates are polysaccharides that may be used as viscosifiers and gel or film-forming agents with a great diversity of applications. The alginates produced by bacteria such as Azotobacter vinelandii are acetylated. The presence of acetyl groups in this type of alginate increases its solubility, viscosity, and swelling capability. The aim of this study was to evaluate, in glucose-limited chemostat cultivations of A. vinelandii ATCC9046, the influence of dissolved oxygen tension (DO) and specific growth rate (μ) on the degree of acetylation of alginates produced by this bacterium. In glucose-limited chemostat cultivations, the degree of alginate acetylation was evaluated under two conditions of DO (1 and 9 %) and for a range of specific growth rates (0.02-0.15 h⁻¹). In addition, the alginate yields and PHB production were evaluated. High DO in the culture resulted in a high degree of alginate acetylation, reaching a maximum acetylation degree of 6.88 % at 9 % DO. In contrast, the increment of μ had a negative effect on the production and acetylation of the polymer. It was found that at high DO (9 %) and low μ, there was a reduction of the respiration rate, and the PHB accumulation was negligible, suggesting that the flux of acetyl-CoA (the acetyl donor) was diverted to alginate acetylation.

Background: The oxygen transfer rate (OTR) and dissolved oxygen tension (DOT) play an important role in determining alginate production and its composition; however, no systematic study has been reported about the independent influence of the OTR and DOT. In this paper, we report a study about alginate production and the evolution of the molecular mass of the polymer produced by a wild-type A. vinelandii strain ATCC 9046, in terms of the maximum oxygen transfer rate (OTRmax) in cultures where the dissolved oxygen tension (DOT) was kept constant., Results: The results revealed that in the two dissolved oxygen conditions evaluated, strictly controlled by gas blending at 0.5 and 5% DOT, an increase in the agitation rate (from 300 to 700 rpm) caused a significant increase in the OTRmax (from 17 to 100 mmol L(-1) h(-1) for DOT of 5% and from 6 to 70 mmol L(-1) h(-1) for DOT of 0.5%). This increase in the OTRmax improved alginate production, as well as the specific alginate production rate (SAPR), reaching a maximal alginate concentration of 3.1 g L(-1) and a SAPR of 0.031 g(alg) g(biom)(-1) h(-1) in the cultures at OTRmax of 100 mmol L(-1) h(-1). In contrast, the mean molecular mass (MMM) of the alginate isolated from cultures developed under non-oxygen limited conditions increased by decreasing the OTRmax, reaching a maximal of 550 kDa at an OTRmax of 17 mmol L(-1) h(-1). However, in the cultures developed under oxygen limitation (0.5% DOT), the MMM of the polymer was practically the same (around 200 kDa) at 300 and 700 rpm, and this remained constant throughout the cultivation., Conclusions: Overall, our results showed that under oxygen-limited and non oxygen-limited conditions, alginate production and its molecular mass are linked to the OTRmax, independently of the DOT of the culture.

The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTR(max)) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTR(max) decreased. The molecular mass obtained at 3.0 mmol l(-1) h(-1) was 7.0 times higher (1,560 kDa) than at 9.0 mmol l(-1) h(-1) (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.

In order to evaluate the independent effects of hydrodynamic conditions and/or oxygen tension on culture physiology and productivity, a fermentation system designed to control dissolved oxygen at constant power drawn (P/V) was developed. The system included a fully instrumented 14 l bioreactor coupled to a PC for data acquisition and control. Power drawn was measured (using a commercial torquemeter coupled to the shaft) and maintained constant by varying the agitation speed; while gas blending was used to control dissolved oxygen concentration. To validate the system, rheological-complex fermentations involving xanthan gum production and filamentous fungal cultivation (using Xanthomonas campestris and Trichoderma harzianum) were developed. In both cases, and despite the changing environmental conditions (due to increased broth viscosities and microbial respiration), both variables were controlled at the desired set points. Through such a system, a rigorous evaluation of the hydrodynamic conditions and/or oxygen tension on culture physiology and productivity is now feasible.

The sizes of air bubbles and castor oil drops were studied by image analysis as a function of the concentration of soluble protein (bovine serum albumin [BSA] and lipase, as model proteins) in a three-phase system using a simulated fermentation medium (aqueous salt solution, castor oil, and air). Small amounts of proteins (<0.02 g/L) caused an important decrease in oil drops and bubbles sizes, together with a pronounced decrease in surface tension. The extent and profiles of this decrease seem to be determined by the conformation of the protein at the interface. The kLa value increased considerably for increasing concentration (up to 0.02 g/L) of the two proteins but was very different (2-fold higher for the lipase) at the highest concentrations tested (0.5 g/L), a phenomenon that can be caused by the extent to which bubbles are trapped within oil drops.

An Azotobacter vinelandii mutant lacking alginate-lyase (SML2) and the wild type (ATCC 9046) were used to discriminate between the roles of the polymerase complex and alginate-lyase in the synthesis of alginate in cultures conducted under controlled dissolved oxygen tension (DOT). To avoid the presence of pre-synthesized alginates, all cultures were inoculated with washed cells. For cultures carried out at 3% DOT using the mutant, a well defined family of alginates of high mean molecular weight (MMW) were obtained (985 kDa). Under 1% and 5% DOT, the mutant produced unique families of alginates with lower MMW (150 and 388 kDa). A similar behavior was observed using the wild type: a production of well defined families of alginates of high MMW at 3% DOT (1,250 kDa) and lower MMW at 1% and 5% DOT (370 and 350 kDa). At the end of the ATCC 9046 fermentations, alginate was depolymerized by the action of lyases. Overall, the evidence indicated that polymerization of alginate is carried out by producing families of polysaccharide in a narrow MMW range, and that it is highly dependent on DOT. The role of alginate-lyase (present in the wild type) is restricted to a post-polymerization step.

The effect of oscillating dissolved oxygen tension (DOT) on the metabolism of an exopolysaccharide-producing bacteria (Azotobacter vinelandii) was investigated, particularly on the mean molecular weight (MMW) of the alginate produced. Sinusoidal DOT oscillations were attained by manipulating the oxygen and nitrogen partial pressures at the inlet of a 1.0 L working volume bioreactor. Periods of 1200, 2400, and 4000 s and average amplitudes between 1.0% and 2.2% DOT, with an oscillation axis fixed at 3% DOT, were tested. A culture carried out at constant 3% DOT was used as comparison. The average wave amplitude had an important effect on the maximum mean molecular weight (MMW(max)) of the alginate produced. The higher the amplitude, the lower the MMW(max). As the average wave amplitudes decreased from 2.2% to 1.0%, the MMW(max) increased from 64 to 240 KDa, respectively. Furthermore, at 3% constant DOT (0.0% of amplitude), a MMW(max) of 350 KDa was obtained. No important effect of the oscillating DOT on kinetics of biomass growth, alginate production, and sucrose consumption was observed, compared with constant DOT. The findings of this study point out that accurate DOT control is crucial if a particular molecular weight species of alginate needs to be produced, particularly in large fermentors, where bacteria are exposed to an oscillatory environment as a result of DOT gradients caused by the high viscosity of the broth and insufficient mixing.

Abstract: The objective of this study was to evaluate the influence of the specific power input and oxygen transfer rate (OTR) on changes in the alginate quality, in terms of the viscosifying power, molecular mass and degree of acetylation, of the polymer produced by Azotobacter vinelandii in shake flasks. Decreasing the shaking frequency from 200 to 100rpm decreased the maximal volumetric power input (P/V)max from a maximal value of 1.5kWm−3 to a minimal value of 0.23kWm−3. As a result of the decrease in the maximal volumetric power input, the maximum oxygen transfer rate (OTRmax) decreased from 6mmolL−1 h−1 to 2.6mmolL−1 h−1 and the RQ (respiratory quotient) increased from 0.95 to 1.21 for the lower and higher power input, respectively. A lower RQ value was associated with the highest conversion value of carbon source (sucrose) to alginate. In contrast, at lower OTRmax values, culture broths with a higher viscosity were obtained. Thus, for the same alginate concentration (∼3.0gL−1), the viscosity of cultures at an OTRmax value of 2.26mmolL−1 h−1 was twice as high with respect to the viscosity observed in the culture broth obtained at a higher power input (OTRmax =6.0mmolL−1 h−1). In addition, under lower power input (lower OTR), the molecular mass and the degree of acetylation of the polymer were the highest. Additional experiments in a stirred bioreactor under constant DOT and agitation rate showed that under non-oxygen-limited conditions (5% of DOT), the alginate yield and the molecular mass of the polymer are determined by the OTR and are independent of the agitation rate of the culture. [ABSTRACT FROM AUTHOR]

Two new dimensionless parameters (Φ and Ω) are proposed for calculating the proportional, integral, and derivative constants of a dissolved oxygen proportional integral-derivative (PID) feed-back control algorithm from knowledge of the growth rate, bioreactor design and operation variables. The values of Φ and Ω were determined for a broad range of Reynolds numbers (between 1000 to 40 000) during the exponential growth phase of two highly different processes: fermentations of recombinant Escherichia coli and cultures of human hematopoietic cells. The utility of Φ and Ω for use in dissolved oxygen self-tunning adaptive control algorithms is discussed. [ABSTRACT FROM AUTHOR]

Abstract: By simulating the evolution of the actual power input observed in shake flasks it was possible to produce, in a fermentor, alginates having a very similar mean molecular mass (1700kDa) to that obtained in the cultures developed in shake flasks (1800kDa). Similar profiles of dissolved oxygen tension and oxygen transfer rate could be the reason. [Copyright &y& Elsevier]