◆ SpookStack

United States Patent: 6,387,665 Page 5 of 11 e © ; » Fermentation conditions: The fermentations described here were carried out using a New Brunswick Bio-Flo 3000 equipped with a 5.0 liter working volume glass vessel and stainless steel headplate and hemispherical bottom cooling dish. Four liters of FA medium were added to the vessel, which had been previously completely disassembled, scrubbed in a dilute Envirochem solution and autoclaved for 15 min after the addition of 4 liters of H2O. The polarographic DO.sub.2 probe (Ingold) and pH probes (either liquid or gel filled, Ingold) were also inserted and all addition and sampling ports were sealed or clamped and wrapped in aluminum foil. Addition lines consisted of surgical grade autoclavable Tygon tubing (Thomas Scientific) and all lines were sealed with the exception of the condenser, which was left open to permit pressure release, but covered with aluminum foil. The vessel was autoclaved using a 10 min exposure time at 121.degree. C. and removed from the autoclave as soon as sufficient cooling had occurred to allow opening of the autoclave. The vessel was then immediately connected to the fermentor unit and the condenser line was connected to a sterile liquid trap and 0.2.mu. capsule filter to avoid the introduction of contaminants during the cooling process. The vessel was then cooled to 37.degree. C. using the fermentor driven temperature control and positive pressure was provided using compressed sterile filtered air. Once the vessel had cooled to 37.degree. C. sterile filtered kanamycin was added to a final concentration of 40 .mu.g/ml. The agitation was activated at 150 rpm and aeration was adjusted to 1-1.2 volume/volume/min (vvm) and antifoam C (DOW), that had been diluted 10-fold into H.sub.2 O and autoclaved, was added to a final concentration of 200 ppm. A preinoculation sterility check was conducted for a minimum of 16 hrs during which time pH, agitation and temperature were continually monitored. After the 16 hrs required for DO.sub.2 probe polarization, the DO.sub.2 was also monitored along with turbidity. The D.sub.2 probe was calibrated using an INGOLD calibration device which sets the zero value to 4 mA and 100% to the oxygen tension determined by the solubility of oxygen in the medium after aeration and agitation at 37.degree. C. The calibration and response of the electrode was then checked by sparging with pure N.sub.2. The vessel was judged to be sterile if the pH and DO.sub.2 remained constant and no increase in turbidity was observed. It should be emphasized that the short autoclave cycle for vessel sterilization was required to minimize caramelization, Millard and other chemical degradation reactions which are problematic due to the high concentrations of yeast extract and tryptone in FA medium. As an additional confirmation of sterility, 50 mls was aseptically removed from the fermentor to a 250 mls Erlenmeyer and incubated at 37.degree, C. at 200 rpm for 48 hrs with no sign of growth. Under the conditions outlined here contamination has not been observed in more than 10 fermentation cycles. Once the sterility of the vessel had been verified, the 300 ml inoculum described above was added to the vessel through the addition port of the headplate and the initial O.D..sub.600nm was recorded. A sample of the inoculum was also streaked on SBA plates and incubated for 48 hrs at 37.degree. C. to verify . inoculum purity. Using the Bio-Flo 3000, aeration was maintained at 75% of saturation by increasing agitation from the initial 150 rpm to a maximum of 400 rpm and ultimately by supplementing the 1 vvm aeration rate with pure oxygen. The mixture rate and percentages of air and oxygen were controlled by a solenoid and algorithm developed by Nev Brunswick Scientific. Both gases had a working pressure of approximately 10 psi. 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.600 determination, two appropriate dilutions were made and results were considered acceptable only when both dilutions yielded a linear response. DCWs 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 hitp://patft_uspto.gov/netacgi/nph-Parser? Sectl=PTO2&Sect2=HITOFF &u=/netahtml/sear... 6/28/2005