(451d) Characterization of Oxygen Availability Measured As Oxygen Transfer RATE and Dissolved Oxygen Tension in Shake Flasks for the Production of O-Mannosylated Proteins in Streptomyces Lividans

Introduction. Shake flask are the most frequently used reaction vessels in biotechnology (Buchs, 2001). In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. Oxygen transfer rate (OTR) is one of the most suitable measurable parameter to quantify the physiological state of an aerobic culture, since most metabolic activities depend on oxygen consumption (Buchs, 2001). The effect of three different shear and aeration conditions using conventional normal (NF), baffled (BF) and coiled (CF) shake flasks on Streptomyces lividans cultures on OTR was evaluated online. Also, the dissolved oxygen tension (DOT) was measured on the three flasks designs. Previous reports of our group indicates that different flask designs in S. lividans cultures leads to changes in O-mannosylation and production of a recombinant protein (Gamboa-Suasnavart et al., 2011), suggesting that oxygen transfer rates are also affected.

Methods. S.lividans cultures were performed at 30ºC, 150 rpm, 60 h in 34% sucrose Luria- Bertani medium (Gamboa-Suasnavart et al., 2011). Corynobacterium glutamicum cultures in same conditions were carried as a higher respiration activity microorganism for comparison. OTR was measured using the RAMOS (Anderlei and Buchs, 2004). This device analyzes the depletion of oxygen concentration in the gas headspace of the shake flask with an oxygen sensor. The slope of this depletion corresponds to the OTR (Anderlei and Buchs, 2004). Carbon transfer rate (CTR) was calculated as the carbon dioxide formation, measured with a sensor in the flask headspace. Three coiled, three baffled and two conventional flasks were used each time. RQ was calculated as the ratio between CTR/OTR. DOT was measured by a luminescent patch (Pst3) using the system Fibox3 by PreSens.

Results and Discussion. Cultures carried out in CF and BF reached a maximum OTR of 9.16 ± 0.15 and 9.36 ± 0.28 mmol/lh respectively after 40 hrs of culture. In contrast, a significantly lower OTR was reached (0.66 ± 0.48 mmol/lh) in those cultures in NF. C. glutamicum cultures presented the same trend, achieving in CF, BF, and NF, 10 ± 0.22, 9 ± 0.31 and 2 ± 0.17 mmol/lh, respectively. RQ values was closer to one in BF and CF, indicating an efficient aerobic metabolism. But in NF, RQ values fluctuates between one and two, suggesting an inefficient carbon oxidation and probably used in other  metabolism pathways.

DOT in BF and CF decrease up to 50% at the end of the culture but in NF a total depletion of oxygen was observed. This might be due to the poor OTR observed in NF cultures compared to BF and CF. On the other hand,  the pellet sizes of S. lividans in NF are seven times (1.57 ± 0.41) higher than those in BF and CF (0.23 ± 0.06 and 0.16 ± 0.05) (Gamboa-S et al. 2011).  By using the equation reported by Cui et al. (1997), an oxygen transfer limitation can be assumed by the higher dissolved oxygen tension needed to diffuse to the center of the pellet (170% DOT).  In the case of the pellets from BF and CF, just 1.76% of DOT is required, demonstrating that in those geometries no oxygen limitations can occur.

Conclusions. Flask design modifies the OTR in microorganism cultures with low and high respiration activity. Higher OTR values are reached when baffles or a spring were used, due to the increase in mass transfer. OTR changes lead to a differences in metabolism as is shown in RQ values. DOT in conventional flask is insufficient to achieve a properly diffusion into pellets in almost whole culture. These OTR differences could lead to changes in recombinant glycoprotein production in S. lividans cultures. The knowledge about the oxygen transfer aspects inside flasks can provide new tools for the development and scale-up of biotechnology processes, mainly in those producing bacterial recombinant glycoproteins.

Acknowledgements. CONACYT-INNOVAPYME 181895, CONACYT 178528, 104951-Z y PAPPIT-UNAM IN-209113, IN-210013

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