◆ SpookStack

United States Patent: 6,316,006 2 aeration rate with pure oxygen. The mixture rate and percentages of air and oxygen were controlled by a solenoid and algorithm developed by New Brunswick Scientific. Both gases had a working pressure of approximately 10 psi. Page 5 of 10 The O.D..sub.600nm dry cell weight (DCW), production of PA, DO.sub.2, pH, agitation and temperature were monitored throughout each fermentation cycle. The O.D..sub.600nm DCW and PA production analysis were carried out by manually sampling the fermentation liquor at hourly intervals using a sterile sampling port. O.D..sub.600nm was measured after dilution of the culture using sterile medium prepared for that fermentation. For each O.D..sub.600nm determination, two appropriate dilutions were made and results were considered acceptable only when both dilutions yielded a linear response. DC Ws were determined starting with a 2 hr point by centrifuging 10 mls of fermentation liquor at 11,953 times.g for 10 min, resuspending the cell pellet in 10 mls of sterile PBS and pelleting the cells again under the same conditions. The cell pellet was resuspended in a minimal volume of PBS and transferred quantitatively to a preweighted Eppendorf centrifuge tube and centrifuged at 14,000 rpm for 5 min. Excess PBS was removed and the cell pellet was dried in a speed-vac for 72 hrs under vacuum and a medium heat setting. A final analysis of the dry weight versus O.D..sub.600nm revealed that the relationship between the two parameters was adequately fit with a linear function. Fermentation Reproducibility: The reproducibility of the cell growth parameters, biomass and PA production in fermentations carried out with the Bio-Flo 3000 under the conditions described above have been summarized in Table I below. Two fermentations were carried out at 75% of the maximum dissolved oxygen concentration in a strict batch mode with no pH control or additions other than antifoam C. The variation in the agitation rate during the first 100 min of the fermentation was the result of the AGDO.sub.2 (agitation DO.sub.2) control mode chosen to maintain the dissolved oxygen tension at 75% of the maximum. Briefly, this algorithm attempts to control the oxygen tension by first altering the agitation rate until this proves insufficient, at which point the process air is supplemented with pure oxygen as needed to maintain the desired DO.sub.2. The temperature was held constant at 37.degree.+/- 0,1.degree. C. The pH was monitored, but not regulated as an internal check on the aeration of the vessel during the course of the fermentation. The fact that the pH revealed a decrease on only 0.2 pH units in the first 150 min was consistent with an aerobic culture metabolizing the limited carbohydrate supplied with the yeast extract to CO.sub.2 and organic acids. Once the carbohydrate was exhausted after ca. 150 min, the bacillus switched to the utilization of amino acids and peptides for a carbon source, which under aerobic conditions resulted in the release of NH.sub.4 OH and the observed increased culture pH. These fermentations were sampled on an hourly basis and allowed to proceed until no further increase in O.D..sub.600nm was observed over two time points. O.D..sub.600nm , DCW analysis and product measurements were carried out for each sample as described above. Samples for PA production were sterile filtered followed by the addition of HEPES and the complete protease cocktail as described under PA quantitation. The samples were concentrated, desalted and ultimately concentrated 80-fold prior to being analyzed using SDS-PAGE. The major band of the gel corresponded to the 83 kDa PA product. An increasing in the intensity of the protein band was seen with increasing fermentation time. Study of a Western blot of another time course of a batch fermentation was developed with polyclonal rabbit anti- PA83. Comparison revealed that along with increasing PA 83 kDa there was also a pronounced increase in the abundance and form of proteolytic degradation products of PA. TABLE 1 Summary of Aerobic .DELTA.Sterne-1 (pPA102)CR4 Fermentations Fermentation Final Conc. Final Yield Final Yield Specific Growth Doubling Time Conditions (.mu.g PA83/ml (mg PA83) (mg PA83/g DCW) Rate T.sub.D (min) Aerobic, Batch 51 235 8.10 0.0132 http://patft.uspto.gov/netacgi/nph-Parser?Sectl =PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005