Multi-stage high cell continuous fermentation for high productivity and titer.

We carried out the first simulation on multi-stage continuous high cell density culture (MSC-HCDC) to show that the MSC-HCDC can achieve batch/fed-batch product titer with much higher productivity to the fed-batch productivity using published fermentation kinetics of lactic acid, penicillin and ethanol. The system under consideration consists of n-serially connected continuous stirred-tank reactors (CSTRs) with either hollow fiber cell recycling or cell immobilization for high cell-density culture. In each CSTR substrate supply and product removal are possible. Penicillin production is severely limited by glucose metabolite repression that requires multi-CSTR glucose feeding. An 8-stage C-HCDC lactic acid fermentation resulted in 212.9 g/L of titer and 10.6 g/L/h of productivity, corresponding to 101 and 429% of the comparable lactic acid fed-batch, respectively. The penicillin production model predicted 149% (0.085 g/L/h) of productivity in 8-stage C-HCDC with 40 g/L of cell density and 289% of productivity (0.165 g/L/h) in 7-stage C-HCDC with 60 g/L of cell density compared with referring batch cultivations. A 2-stage C-HCDC ethanol experimental run showed 107% titer and 257% productivity of the batch system having 88.8 g/L of titer and 3.7 g/L/h of productivity. MSC-HCDC can give much higher productivity than batch/fed-batch system, and yield a several percentage higher titer as well. The productivity ratio of MSC-HCDC over batch/fed-batch system is given as a multiplication of system dilution rate of MSC-HCDC and cycle time of batch/fed-batch system. We suggest MSC-HCDC as a new production platform for various fermentation products including monoclonal antibody.

Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen.

Fed-batch cultures of Hansenula polymorpha were studied to develop an efficient biosystem to produce recombinant human serum albumin (HSA). To comply with this purpose, we used high purity oxygen supplying strategy to increase viable cell density in a bioreactor and enhance the production of target protein. A mutant strain, H. polymorpha GOT7 was utilized in this study as a host strain in both 5-L and 30-L scale fermentors. To supply high purity oxygen into a bioreactor, nearly 100 % high purity oxygen from commercial bomb or higher than 93 % oxygen available in-situ from a pressure swing adsorption oxygen generator (PSA) was employed. Under the optimal fermentation of H. polymorpha with high purity oxygen, the final cell densities and produced HSA concentrations were 24.6 g/L and 5.1 g/L in the 5-L fermentor, and 24.8 g/L and 4.5 g/L in the 30-L fermentor, respectively. These were about 2-10 times higher than those obtained in air-based fed-batch fermentations. The discrepancies between the 5-L and 30-L fermentors with air supply were presumably due to the higher contribution of surface aeration over submerged aeration in the 5-L fermentor. This study, therefore, proved the positive effect of high purity oxygen to enhance viable cell density as well as target recombinant protein production in microbial fermentations.

Rapid separation of bacteriorhodopsin using a laminar-flow extraction system in a microfluidic device.

A protein separation technology using the microfluidic device was developed for the more rapid and effective analysis of target protein. This microfluidic separation system was carried out using the aqueous two-phase system (ATPS) and the ionic liquid two-phase system (ILTPS) for purification method of the protein sample, and the three-flow desalting system was used for the removal of salts from the sucrose-rich sample. Partitioning of the protein sample was observed in ATPS or ILTPS with the various pHs. The microdialysis system was applied to remove small molecules, such as sucrose and salts in the microfluidic channel with the different flow rates of buffer phase. A complex purification method, which combines microdialysis and ATPS or ILTPS, was carried out for the effective purification of bacteriorhodopsin (BR) from the purple membrane of Halobacterium salinarium, which was then analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and matrix-assisted laser desorptionionization time-of-flight. Furthermore, we were able to make a stable three-phase flow controlling the flow rate in the microfluidic channel. Our complex purification methods were successful in purifying and recovering the BR to its required value.

Anaerobic organic acid production of food waste in once-a-day feeding and drawing-off bioreactor.

Acidogenesis of food waste was studied in a 2-L reactor with semi-continuous mode operation (once-a-day feeding and draw-off) for maximum 65 days to examine optimal volatile acid compositions for biological nitrogen removal (BNR) and enhanced biological phosphorus removal (ENPR). Various operational parameters of hydraulic retention time (HRT), organic loading rate (ORL), pH and temperature were investigated for soluble chemical oxygen demand (SCOD), volatile fatty acid composition, nitrogen and phosphate. The yields (gTVFA/g VS) and the volumetric productivity (gTVFA/d L) increased with HRT from 0.26-0.32, 1.25-1.50 (at 4 days) to 0.36-0.39, 1.71-1.83 (at 12 days). However, the acetate fraction (%) decreased with HRT from 35.7-37.5 at 4 days to 23.5-25 at 12 days. The yields decreased with increase of organic loading from 0.34-0.37 at 5 g/L d to 0.29-0.30 at 13 g/L d and the productivity increased from 1.63-1.65 to 3.61-3.75. The yield and productivity were highest at 35 degrees C among 25, 35 and 45 degrees C. The yield and productivity at pH 5.5 and 6.0 were best and very similar to each other. The condition of 35 degrees C, pH 6.0, HRT 8 days, ORL 9 g/L d resulted in TVFA, SCOD, acetate and butyrate of 25, 39.5, 12 and 5.25 g/L, respectively.