Results obtained in our laboratory showed that cell lines of Beta vulgaris (Rodríguez-Monroy and Galindo, 1999) and Uncaria tomentosa (Trejo-Tapia et al, 2007) may be cultivated in shake flasks plugged with aluminum foil. In contrast, the culture of A. indica continuously loses its viability and reduces its pH in these conditions, and it is necessary regenerate periodically new suspension cultures from callus.
In order to know the oxygen demand by each specie, it was used a stirred tank bioreactor stirring with a 45º inclined - 6 blades impeller (400 rpm) and 0.08 vvm of aeration. Oxygen was measured using a polarographic electrode. Cell suspension cultures produced in shake flasks were used to inoculate the biorreactor. OUR was determined by the dynamic method (Doran, 1995). The measurements were done in the first hour after inoculation and the cells were subjected to conditions without oxygen limitation (oxygen dissolved higher than 30 %). OUR was calculated considering the variation of oxygen dissolved with the time, and the biomass concentration present in the bioreactor. The values of OUR obtained were: A. indica 0.100, B. vulgaris 0.027 and Uncaria tomentosa 0.026 kg O2/(kg dry cells.day). Those results indicate A. indica cells consume 4.5 times more oxygen than B. vulgaris or U. tomentosa.
On the other hand, OTR of shake flasks was determined using different plugs, aluminum foil and cotton. So, the overall oxygen mass transfer coefficient (Ko) and the maximum OTR (OTRmax) of the flasks were measured in 500 mL shake flasks at 120 rpm with 100 mL of culture medium, using the dynamic gassing out method. It can be demonstrate that Ko = kplug.kLa.H/(kplug.H + kLa), being kplug the oxygen transfer coefficient through the plug, kLa the oxygen transfer coefficient through the gas-liquid interface and H, the constant of Henry law. Besides, OTRmax = Ko.CO2*, being CO2* the oxygen concentration in the liquid phase at equilibrium with air at the system temperature.
Ko was 0.42 and 3.51 h-1 and OTRmax was 0.07 and 0.58 kg O2/(m3.day) for aluminum and cotton closure respectively. The values of kLa and kplug showed that the main resistance to oxygen flow was in the plug and not in the interface liquid gas, using both kinds of closure. With the values of OTRmax, it is possible to estimate the maximum OUR for a culture with 10 kg dry cells/m3 in shake flasks. The cultures could have a maximum OUR in shake flask of 0.007 and 0.058 kg O2/(kg dry cells.day) for aluminum and cotton closure respectively. Comparing the theoretical OUR obtained in shake flask with those measured in bioreactor for the three species (0.026-0.100 kg O2/(kg dry cells.day)), it is possible to observe that the measured values were higher than some values of maximum OUR in shake flask. This result indicated that the cultures of the three species cultures in shake flasks could have oxygen limitation.
Using oxygen balance is possible to show that A. indica cells have to reduce their OUR below 0.100 kg O2/(kg dry cells.day) when they reach a biomass concentration of 0.7 and 5.7 g DW/L in shake flasks plugged with aluminum and cotton respectively. These results suggests that the cell culture consuming an high quantity of oxygen (A. indica) could no adapt to oxygen limiting condition with foil aluminum, and the products of fermentation under anaerobic conditions such as lactic acid and ethanol, could be a cause of a reduction in pH. On the other hand, U. tomentosa cells have to reduce their OUR below 0.026 kg O2/(kg dry cells.day) when they reache a biomass concentration of 2.7 and 22.1 g DW/L in shake flasks plugged with aluminum and cotton respectively
With the purpose of studying the changes of pH and viability during the subcultures of A. indica, cells of this specie were grown in shake flasks plugged with aluminum foil and cotton. Cell viability and pH were evaluated during 6 continuous subcultures. Results obtained with shake flasks plugged with aluminum foil showed that pH decreased from 5.1 to values below 4.0, while in flasks with cotton, pH was above 5.0. Cell viability decreased from 78 % to 16 % in flasks with aluminum, while the cultures in flasks plugged with cotton had viability above 70 %. Cell suspension cultures with pH below 4.0 stopped their cell growth. A. indica cultures obtained under these conditions presented evident differences in their morphology. Cells cultured in flasks with aluminum foil are composed by aggregates with a diameter lower than 1 mm, while the agglomerates growing in flasks plugged with cotton had a diameter between 3 and 10 mm. Possibly a smaller size of agglomerates facilitates a better oxygen transfer under conditions with too little oxygen.
The results obtained in this work are in agree with those reported by Geigenberger (2003), who showed that plant tissues can reduce the oxygen consume when external concentration oxygen falls o when internal oxygen concentration falls into the tissue even in well oxygenated atmospheres. The plants can reduce the respiration, biosynthetic process, protein degradation or modify their morphology to avoid internal anoxia, and only with oxygen concentration is near to zero, they activate the lactate dehydrogenase and alcohol dehydrogenase for production of anaerobic ATP.
In conclusion, plant cells cultured in shake flask can have an oxygen limitation, so in flasks closured with aluminum foil, as well flasks closured with cotton. If the OTR offered by the flask is too low, the cells can reduce its viability, especially if the plant specie has a higher oxygen uptake rate, as was the case of A. indica. It is necessary to analyze the oxygen consumption of the different plant species in particular way. So, if the plant cell culture has a high oxygen consumption should be studied the effect of one low OTR in primary and secondary metabolism and its morphology. For increasing the OTR in shake flasks could be necessary modify the closure or others variables affecting the OTR (e.g. volume of culture medium in the flask or agitation speed).
Acknowledgements
F. Orozco – Sánchez thanks to Secretaría de Relaciones Exteriores of México for the doctoral fellowship awarded.
References
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