High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol.

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.

Interaction of transport resistances with biochemical reaction in packed-bed solid-state fermentors: effect of temperature gradients.

In solid-state fermentation, the interaction of transport phenomena with biochemical reactions has a considerable effect on the productivity of the bioreactor. Previous work on solid-state fermentation in tray fermentors in our laboratory indicated that heat transfer resistance results in steep temperature gradients within the solid substrate bed, which in turn adversely affect the biochemical reaction and enzyme activity. This problem of heat accumulation during the course of fermentation has been alleviated to a considerable extent using a packed-column bioreactor with forced aeration in the present work. Experimental studies were conducted in a packed-column bioreactor utilizing wheat bran as substrate and the fungus Aspergillus niger CFTRI 1105 for the production of the enzyme amyloglucosidase. The enzyme activities were estimated and temperatures were recorded at different bed heights, for different air flow rates during the course of fermentation. The results indicated that the temperature gradients caused by heat transfer resistances were reduced considerably with corresponding increases in enzyme activity.

Interaction of transport resistances with biochemical reaction in packed bed solid state fermenters: the effect of gaseous concentration gradients.

Mass transfer plays an important role in solid state fermentation (SSF) systems. Earlier work on SSF in tray bioreactors indicated that steep gaseous concentration gradients developed within the substrate bed, owing to mass transfer resistances, which may adversely affect the bioreactor performance. For all practical purposes these gradients have been eliminated using a packed bed column bioreactor with forced aeration. Gaseous concentrations (oxygen and carbon dioxide) and enzyme activities were measured at various bed heights for various air flow rates during the course of fermentation. The results indicated that concentration gradients were decreased effectively by increasing air flow rate. For example, the actual oxygen and carbon dioxide concentration gradients reduced from 0.07% (v/v) cm-1 and 0.023% (v/v) cm-1 to 0.007% (v/v) cm-1 and 0.0032% (v/v) cm-1 respectively when the air flow rate was increased from 5 dm3 min-1 to 25 dm3 min-1. This resulted in an overall improvement in the performance of the bioreactor in terms of enzyme production.

Gas concentration and temperature gradients in a packed bed solid-state fermentor.

In solid-state fermentation (SSF), interaction of heat and mass transfer with biochemical reaction (growth associated enzyme production) affects the bioreactor performance. This interaction was earlier observed to cause temperature and gaseous concentration gradients which reduced the effective bed height of the bioreactor. Since forced aeration is known to alleviate this problem, a packed column bioreactor with forced aeration was employed in the present study. Using wheat bran and Aspergillus niger CFTRI 1105, experiments were conducted for the production of the enzyme amyloglucosidase at various air flow rates. Temperatures and gas concentrations were recorded and enzyme activities estimated at different bed heights during the course of SSF. Gas concentration and temperature gradients decreased with increasing air flow rate. The packed column allowed the use of larger bed heights and yielded higher enzyme activities (6,260 Units/gDMB) than trays (345 Units/gDMB). Enzyme activity was affected more by temperature than concentration gradients, and increased with air flow rates.

Cultural stability of Streptomyces fradiae in the production of xylose isomerase: studies in shake flasks.

Comprehensive studies of pure colonies of Streptomyces fradiae in the production of xylose isomerase by submerged fermentation at shake flask level revealed poor culture stability with respect to enzyme production, biomass formation, degree of pigmentation, quantity of glucose and xylose utilization, level of enzyme in cell-free culture broth and final pH of the fermentation medium. The results serve to stress obligatory evaluation of culture stability of Streptomyces strains in determining their suitability for use in developing fermentation processes for commercial exploitation.