Recombinant Pichia pastoris is one of the important methylotropic yeast due to its robustness and ability to produce hormones like human chorionic gonadotropin (hCG), luteinizing hormone (LH) extracellularly. High growth on glycerol and strong protein expression on methanol by insertion of alcohol oxidase (AOX) promoter demand the fermentation to be a multistage operation. Methylotropic pathway demands more oxygen as methanol has to be converted to formaldehyde with half mole of oxygen. Moreover as fermentation progresses cell density in the reactor also increases. In case of Pichia pastoris fermentation cell density usually reaches very high (above 100 gm/lit) at the end of fermentation. Both these contribute in
the increased oxygen demand in the fermentation and oxygen transfer turns out to
be a limiting step. The present study focuses on the oxygen transfer process and its improvement in the fermentation.
Oxygen transfer in bioreactor is a multistep process and involves different kinetic as well as mass transfer steps. In case of fermentation especially at high cell
densities, oxygen transfer from bubbles to the broth becomes limiting step. The
interface transport is governed by many physical as well as kinetic parameters. It is
essential to screen these parameters from the whole set to identify the key parameters. Sensitivity analysis is carried out by using Metabolic Control Analysis (MCA)
to quantify the effects of different parameters. It is found that bubble size and oxygen partial pressure are two such key parameters which can be manipulated. Use of
pure oxygen to increase partial pressure and thereby solubility of oxygen in broth
is a common approach. This work focuses on bubble size manipulation to increase the oxygen transfer rates.The idea behind this work is on to generate micron sized bubbles and utilize them effectively in the fermentation.
There are many techniques reported to generate microbubble dispersions. In this
work ’Spinning Disc microbubble Generator’ is fabricated to generate microbubbles. A flat disc surrounded by baffles with 5 mm gap in between, when subjected to 5000 rpm generates microbubbles. Some modifications are done to the set up to achieve desired properties of the bubbles. The bubbles generated fall in the range of 30-300 micron with mean size of about 60 micron. Use of Tween-20 surfactant stabilize the bubbles and hence offer a good resistance to coalescence and breakage. The liquid fraction in the bubbles can be as high as 40%. Contineous addition of this dispersion unnecessarily can dilute the fermentation broth. To overcome this volume constrain, a recirculation system is designed. Microbubble dispersion is added contineously to the reactor and equivalent fermentation broth is pumped back to the microbubble generator to achieve steady state to the liquid volume in both the vessels.
Mass transfer studies with microbubbles show the potential of microbubble dispersion (MBD) to enhance mass transfer significantly. Decrease in volumetric mass
transfer coefficient (KLa) due to surfactant is overcompensated by the increase in
the interfacial area and net effect is, potential enhancement in KLa. The enhance-
ment factor, that is, ratio of mass transfer coefficient with MBD to mass transfer
coefficient with conventional sparging, is obtained to be about 4 to 5. Prior to
utilization of bubbles in the recirculation system, cells are checked for the shear
sensitiveness. Negligible lysis losses and almost no effect on growth patterns in
shake flask culture confirm that the cells used are mechanically stable at operating
conditions.
Better growth patterns in shake flask are observed when microbubbles are pumped for predetermined duration in the broth. It shows possible use of MBD as oxygen carriers. Glycerol batch phase with MBD and conventional sparging is studied at
different initial cell densities. Conventional sparging fails to grow the cells and Dissolved Oxygen (DO) levels close to zero suggest high oxygen demands which
can not be sustained by conventional sparging. The same batch is run using MBD.
Reasonably good growth patterns are observed. DO levels are well above 70% for
most of the time during operation. High oxygen demand which can not be sustained
by conventional sparging alone can be sustained by MBD. In this way in high den-
sity cultures utilization of MBD can be a good alternative to fulfill required oxygen
demand in fermentation.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/432 |
| Date | 09 1900 |
| Creators | Subhash, Kaujalgikar Saurabh |
| Contributors | Modak, Jayant M |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G20537 |
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