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Friday, April 1, 2011



To estimate the volumetric mass transfer coefficient (KLa) by dynamic gassing method.

            Gas liquid mass transfer is of paramount importance in bioprocess because of the requirement of oxygen in aerobic fermentations. One of the most critical factors in the operation of a fermentor is the provision of adequate gas exchange. Oxygen is the most important gaseous substrate  for microbial metabolism and carbon-dioxide is the most important gaseous metabolic product.

            When oxygen is required as a microbial substrate, it is frequently a limiting factor in fermentation. Because of its low solubility, only 0.3 mM oxygen, equivalent to 9 mg/ml, dissolves in one liter of water at 20ºC in an air/ water mixture. Due to the influence of the culture nutrients, the maximal oxygen content is actually lower than it would be in pure water. As temperature increases, solubility of oxygen reduces. For example, at 33ºC the solubility is 7.2 mg/l.
Most aerobic microbial processes are oxygen limited. That is the reason why the concept of gas –liquid mass transfer in bioprocess is centered on oxygen transfer even if other gases such as carbon dioxide and ammonia can be involved.

            The solubility of gases follows Henry’s law in the gas pressure range over which the fermentors are operated. This means that if the oxygen concentration in the gas phase increases, the oxygen proportion of the nutrient solution increases. Consequently the highest oxygen partial pressures are attained during aeration with pure oxygen. Compared to value in air (9 mg/l), pure oxygen has a solubility of 43 mg/l in water.

            For oxygen to be transferred from a gas bubble to an individual cell, several independent partial resistances must be overcome.
·         Diffusion from the bulk gas-to-gas liquid interface.
·         Movement through gas liquid interface.
·         Diffusion of oxygen through relatively unmixed liquid region adjacent to the bubble into the well-mixed bulk liquid.
·         Transport of oxygen through bulk liquid to a second relatively unmixed liquid region surrounding the cells.
·         Transport through second unmixed liquid region associated with the cells.
·         Diffusive transport through cellular floc.
·         Transport across the cell envelope and to intracellular reaction site
The mass transfer of oxygen into liquid can be characterized by oxygen transfer coefficient KLa. This has been thoroughly examined as a critical parameter for bioreactor function. It depends upon the following parameters:
·         Vessel geometry
·         Mixing properties
·         Aeration system
·         Nutrient solution
·         Micro organism
·         Antifoam agent
·         Temperature
Baffles produce a large planer liquid surface and a uniform flow pattern and also increases liquid hold up for a given fermentor volume.
Surfactants such as antifoam agents reduce oxygen transfer rate.
Microorganisms act as a barrier for oxygen transfer and hence reduce oxygen transfer rate.
The gas bubbles are replenished in the locations of the bioreactor where there is negative pressure, such as behind the agitator blades. As aeration increases, various conditions can be characterized.
·         At low aeration rates, large bubbles form behind the turbine blades and smaller bubbles are spun off into the nutrient solution.
·         As the aeration rate is increased, gas bubbles collect behind all the turbine blades and continue to accumulate. The energy input is one-third less than that used for un aerated systems.
·         In this intermediate stirring range, gas dispersion is the best.
·         At every high aeration rates, many large bubbles adhere to each other and the impeller is flooded with gas, resulting in sharply lowered gas dispersion.

            The critical oxygen concentration is the term used to indicate the value of the oxygen uptake rate or oxygen absorption rate which permits respiration without hindrance. Generally the critical oxygen concentrations are 5-25% of the oxygen saturation value in culture. At oxygen absorption rates that are lower than the critical concentrations, respiration rate is correlated with the oxygen concentration in the solution. Above this value, no dependence between respiration rate and dissolved oxygen has been observed. In yeast and bacterial fermentations, the critical oxygen concentration is constant and not affected by fermentation conditions. In filamentous microorganisms, the critical oxygen concentration has been shown to be dependant on fermentation conditions.

The reduction of dissolved oxygen at a noble metal surface negatively polarized with respect to reference electrode forms the basis of design and operation of oxygen electrodes. Membrane covered oxygen probes measure oxygen partial pressures because the electrode generates the current that is proportional to the amount of oxygen through the membrane that is directly proportional to the oxygen tension in the medium.
The two type of oxygen electrodes that may be used are:
·         Polarographic electrodes.
·         Galvanic electrodes.
            A constant potential difference is maintained across a reference electrode (Anode-Calomel or Silver / Silver Chloride) and a platinum cathode in a suitable electrolyte like Potassium chloride. In this design, a membrane that is permeable to oxygen (PTFE or Silicon reinforced with steel mesh) separates the fermentation fluid from the electrode. Oxygen diffuses through the membrane to the cathode where it reacts to produce a current between the anode and the cathode proportional to the oxygen partial pressure in the fermentation broth.
  O2 + 2H2O + 2e-               H2O2 + 2OH-
                                  H2O2 + 2e-              2OH-
 Ag  + Cl-                AgCl  + e-
Overall reaction:

             4Ag  +  O2 + 2H2O + 4Cl-                4AgCl +  4OH-

            The 5l fermentor was aerated by air supplied from tanks through a compressor. Air was passed into the fermentor through a flow meter / regulating valve system and then through a sterilizing filter. It was introduced at the bottom of the filter through a   sparger, which is an arrangement of pipe work perforated with small holes.
By shutting off the regulating valve, the air supply to the fermentor was stopped. The fall in the DO concentration was noted down at regular intervals. After the DO level came to around 20%, the valve was opened to allow inflow of air. The rise in DO concentration was noted at regular intervals till the value become constant.

            This method for measuring KLa is based on unsteady state mass balance for oxygen. At some time, the broth is deoxygenated by stopping the airflow. DO concentration [DO] drops during this period. Air is pumped to the broth at a constant flow rate and increase in [DO] is monitored as a function of time. It is important that the level remains above the critical DO concentration so that the oxygen uptake is independent of oxygen level. Assuming the re-oxygenation of the broth is fast relative to cell growth, the dissolved oxygen level, will soon reach a steady state value [DO] which reflects a balance between oxygen supply and consumption in the system.
            During the cut- off step, there is no mass transfer as the dissolved oxygen is only used. Hence,
                                   QoX = d[DO]/dt
During the re-oxygenation step, the system is not at steady state,

d[DO]/dt = KLa (d[DO] /dt + Qo2X) / ( [DO]* - [DO])   

The obtained KLa value is in the lower range that implies that the ability of the reactor to deliver oxygen to the cells is limited. The Qo2X is lower in the second case. This might be because the cells were subjected to oxygen concentrations below critical levels or might be because of reduction in impeller velocity.

            KLa can be used characterize oxygen mass transfer capability of fermentors. Under steady state there can be no accumulation of oxygen at any location in the fermentor. Therefore, the rate of oxygen transfer from the bubbles must be equal to rate of oxygen consumed by the cells.
                                    KLa ([DO]* -[DO]) = QO2X
We can predict the response of fermentor to change in mass transfer conditions using the above equation

The volumetric mass transfer coefficient (KLa) determined by the dynamic gassing method


·         The KLa obtained (0.006 S-1) quite low. This can be attributed to the following reasons.
·         When the readings were taken the cells were suspected to have entered the death phase. (Since it had been 6hrs from the start of the reactor.)
·         During the gassing phase the KLa is limited by the mass transfer coefficient between air supplied over the medium. It can be eliminated by increased aeration. Because of the increased residence time of O2 bubbles in medium resulting in low KLa value.
·         The response of the probe may not be good. A thorough knowledge of the response time of the probe is essential

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