Fermentation vessels sampling

It is very simple to perform batch fermentation in a small flask with a volume of say 200 ml. Now our target is to use a 2 litre B. Braun fermenter. All accessories are shown in Figure 10.5. The fermentation vessel only, as shown in Figure 10.6, with about 250 ml of media without any accessories but with some silicon tubing attached with a filter for ventilation is autoclaved at a 131 °C for 10 minutes at 15psig.9 After that, the system is handled with special care and all accessories attached. Media is separately sterilised and pumped into the vessel. Inoculum is transferred and the batch experiment is started right after the inoculation of seed culture. An initial sample is withdrawn for analysis. 

A cross-flow nanofillration module (SEPA CFII, GE Osmonics, Miime lis, MN) was used for this process with a maximum operating pressure of 7.0 MPa. The sur ce area of the membrane is 140 cm. The holdup volume of the membrane unit is 70 mL. The fermentation broth was placed in a 5-L fermentation vessel to control the temperature, agitation, and pH. A bench-top pump (M03-S, Hydra cell, MinneapoUs, MN) was used to pump the fermentation broth through the cross-flow membrane separation unit and recycle back to the fermentor (Fig. 2). The permeate was collected on a digital balance attached to a laptop computer with a RS-COM version 2.40 system (A D, Milpitas, CA) that recorded the amount of permeate collected every 0.5 min. The fermentation brofli was kept at constant temperature (37 °C), pH (5.5), and agitation (200 rpm). Transmembrane pressures of 1.4, 2.1, and 2.8 MPa were used in the nanofiltration tests. Each condition was tested twice, and each test lasted for 2 h. Samples of the original broth (before separation), permeate, and letentate were collected for analysis. 

Scale-Up Fermenters ranging from about two to over 100 hters (0.07-3.5 fP) have been used for research and development, but the smaller sizes provide too httle volume for sampling and are difficult to replicate, whue large vessels are expensive and use too much medium. Autoclavable small fermenters that are placed in a water bath for temperature control are less expensive than vessels with jackets or coils, but much labor is required for handling them. Pressure vessels that... 

Addition of materials or cultures to fermenters and other vessels, and the taking of samples, should be carried out under carefully controlled conditions to ensure that absence of contamination is maintained. Care should be taken to ensure that vessels are correctly connected when addition or sampling take place. 

Prior to the fermentations, the pH of the different hydrolysate samples was adjusted to 5.5 with NaOH (5 M). All fermentations were carried out under oxygen-limited conditions in 55-mL glass vessels containing 50 mL of medium of which 47.5 mL was hydrolysate (or, alternatively, an aqueous glucose solution for reference fermentations). The vessels were sealed with rubber stoppers and equipped with cannulas for outlet of C02. The hydrolysates were supplemented with nutrients as previously described (20). Fermentations of 35 g/L of glucose and nutrients but no hydrolysate were used for reference. The flasks were inoculated to an initial cell mass concentration of 2.0 g/L dry wt and incubated at 30°C with stirring. The fermentations were run for 36 h. Samples of 200 pL were taken after 0,2,4, 6, 8,10, 24, and 36 h. 

Aseptic systems are used to transfer the inoculum to the vessel, to allow the removal of routine samples during fermentation, for early harvesting of aliquots when the vessel becomes full as a consequence of the media additions and to transfer the final contents to the extraction plant when fermentation is complete. Asepsis is assured by engineering design and by steam, which must reach all parts of the vessels and associated pipework. Any pockets of air or rough surfaces that steam does not penetrate could act as reservoirs for foreign growth. 

Weise et al. (1987) proposed saturating food and fermentation samples by bubbling with air directly in the reaction vessel of a modified automated sampler. During the oxygenation, hydrolysis of the analytes sucrose, glucosinolate, or starch was performed. Up to 60 samples per hour could be analyzed automatically with good precision. 

Fermenter studies were carried out in a New Brunswick continuous culture apparatus with 15-liter stirred fermenter jars for culture vessel and nutrient reservoirs and a 20-liter carboy for a harvest vessel. Temperature of the culture was maintained at 28°-29°C. Impeller speed was kept at 120 r.p.m. with a 3-inch diameter set of four impeller blades low in the culture, and a second impeller just above the liquid level to aid in foam control. A few drops of Dow Coming Antifoam AF at 25 mg./ml. (emulsion) were added manually as required to control foam. The rate of aeration was 1 to 3 liters per minute. Contamination through the impeller shaft port was prevented by a loose fitting sleeve through which live steam was continually passed (10). Samples taken from the fermenter were streaked on potato dextrose agar, and Bacto plate count agar, incubated for 7-10 days and inspected for contamination. 

Fermenter contaminated[ [inoculum tank contaminated] /inoculum line con-taminated/procedure wrong/tank dirty/air leak/leak from the coil or jacket/faulty sensors/antifoam is not sterile/dirty gaskets, bottom valve, sample line and valve, vent line valve, vacuum breaker/nutrient feed tank or line not sterile/all lines wee not up to sterilization temperature/steam condensate left in lines/the humidity of the fermenter air upstream of the sterile filter is > 90%/pH and DO probes were not deaned between runs/probe holders were not brushed and deaned with a hypochlorite or formaldehyde solution/for a previously contaminated vessel the valves and gaskets were not replaced, instrument sensors were not removed and cleaned high boiling germicide, such as sodium carbonate or sodium phosphate was not used. 

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