The high cell density fermentation of recombinant Pichia pastoris for human serum albumin (HSA) production is a high oxygen demand process. The oxygen demand is usually met by increased agitation rate and use of oxygen-enriched air. Microbubble fermentation however can supply adequate oxygen to the microorganisms at relatively low agitation rates because of improved mass transfer of the microbubbles used for the sparging. Conventionally sparged fermentations were conducted for the production of HSA using P. pastoris at agitation rates of 350, 500, and 750 rpm, and were compared to MBD sparged fermentation at 150, 350, and 500 rpm agitation rates. The MBD improved the volumetric oxygen transfer coefficient (kLa) and subsequently increased the cell mass and protein production compared to conventional fermentation. Cell production in MBD fermentation at 350 rpm was 4.6 times higher than that in conventional fermentation at 350 rpm, but similar to that in the conventional 750 rpm. Maximum cell mass productivity in the conventional 350 rpm was only 0.37 g / (L·h), while the maximum value in MBD 350 rpm was 2.0 g / (L·h), which was similar to 2.2 g / (L·h) in the conventional 750 rpm. Biomass yield on glycerol Ys (g cell/ g glycerol) was 0.334 g / g in the conventional 350 rpm, 0.431 g / g in MBD 350 rpm and 0.438 g / g in the conventional 750 rpm. Protein production in MBD 350 rpm was 7.3 times higher than that in the conventional 350 rpm, but similar to the conventional 750 rpm. Maximum protein productivity in the conventional 350 rpm was 0.37 mg / (L·h), 2.8 mg / (L·h) in MBD 350 rpm, and 3.3 mg / (L·h) in the conventional 750 rpm. Protein yield on methanol Yp (mg protein / g methanol) was 1.57 mg /g in the conventional 350 rpm, 5.02 in MBD 350 rpm, and 5.21 in the conventional 750 rpm. The volumetric oxygen transfer coefficient kLa was 1011.9 h-1 in MBD 350 rpm, which was 6.1 times higher than that in the conventional 350 rpm (164.9 h-1) but was similar to the conventional 750 rpm (1098 h-1). Therefore, MBD fermentation results at low agitation of 350 rpm were similar to those in the conventional fermentation at high agitation of 750 rpm. There was considerable improvement in oxygen transfer to the microorganism using MBD sparging relative to the conventional sparging. Conventional fermentations were conducted both in a Biostat Q fermenter (small) at 500 rpm, 750 rpm, and 1000 rpm, and in a Bioflo III fermenter (large) at 350 rpm, 500 rpm, and 750 rpm. At the same agitation rate of 500 rpm, cell production in the large reactor was 3.8 times higher than that in the small one, and no detectable protein was produced in the small reactor at 500 rpm. At the same agitation rate of 750 rpm, both cell production and protein production in the large reactor were 4.6 times higher than the small reactor. Thus, the Bioflo III fermenter showed higher oxygen transfer efficiency than the Biostat Q fermenter, because of the more efficient aeration design of the Bioflo III fermenter. / Master of Science

In the large-scale production of <i>Saccharomyces cerevisiae</i> (baker's yeast), oxygen transfer, which is one of the major limiting factors, is improved by using high agitation rates. However, high agitation rates subject the microorganisms to high shear stress and caused high power consumption. A microbubble dispersion (MBD) method was investigated to improve oxygen transfer at low agitation rates and thus reduce power consumption and shear stress on the microorganisms. The experiments were conducted at the 1-liter level and subsequently scaled-up to 50-liters using a constant volumetric oxygen transfer coefficient (<i>k_La</i>) method for scaling. In comparison to a conventional air-sparged fermentation, the MBD method considerably improved the cell mass yield, growth rate and power consumption in the 50-liter fermentor. Cell mass production in the MBD system at agitation rate of 150 rpm was about the same as those obtained for a conventional air-sparged system agitatid at 500 rpm. Power consumption in the conventional air-sparged system was three-fold that required for the same biomass yield in the MBD system. However, at the 1-liter scale, the MBD system did not show any significant advantage over the air-sparged system because of the high power consumption. / Master of Science

A microbubble dispersion (MBD) was used to supply oxygen for an aerobic fermentation of Baker's yeast. The 1-liter microbubble dispersion generator supplied bubbles for 20-liter and 50-liter working volume fermentations in a 72-liter pilot scale fermenter. The microbubbles were stabilized by the surfactants naturally present in the culturing broth medium. The growth patterns of yeast Saccharomyces cerevisiae, cultured at agitation speeds of 150 rpm and 500 rpm, were compared for oxygen supplied by ordinary air sparging and by MBD sparging. Both air sparged and MBD systems were supplied air at equivalent volumetric flow rates. The volumetric oxygen transfer coefficients (KLa) were estimated by the yield coefficient method. The KLa values increased from 142.5 to 458.3 h-1 and from 136.1 to 473.3 h-1 for 20- and 50- liter runs, respectively, as the agitation speed was increased from 150 to 500 rpm in the ordinary air sparged fermentations. The oxygen transfer coefficients in the MBD sparged fermentations were found to be independent of the fermenter agitation speed at approximately 480 h-1 for 20-liter runs and 340 h-1 for 50-liter runs. The growth rates for MBD at 150 rpm were essentially equivalent with air sparged fermentations at 500 rpm. The total power consumption per unit volume of broth for the 150 rpm, MBD fermentation was 68% lower than the 500 rpm, air sparged run for the 20-liter fermentations and was 55% lower for the 50-liter fermentations. / Master of Science